Methods of modulating hair growth

ABSTRACT

The invention features methods of promoting hair growth in a subject. The methods include inducing or mimicking the effects of Wnt promoted signal transduction, e.g., by increasing the level of Wnt protein or administering an agent which mimics an effect of Wnt promoted signal transduction, e.g., by administering lithium chloride. Methods of inhibiting hair growth are also provided.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 60/261,690, filed Jan. 12, 2001, and U.S. provisional applicationSer. No. 60/193,771, filed Mar. 31, 2000, both of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

The hair follicle undergoes a cycle of hair growth (anagen) followed byregression (catagen), and quiescence (telogen) until a new hair shaft isgenerated in the existing follicle during the subsequent anagen phase.Hardy et al. (1992) Trends in Genetics 8:55-61. The hair shaft isderived from the epithelial matrix cells at the base of the follicle,but a cluster of dermal cells ensheathed by the matrix cells, known asthe dermal papilla (DP), is thought to supply inductive signals requiredfor hair outgrowth. Reciprocal signaling from the epidermis is requiredfor the formation of the dermal papilla and may also explain thecoordinated morphological changes in epithelial and dermal components ofthe follicle observed during the hair cycle.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery that increasing Wntprotein levels can positively regulate the ability of dermal papilla(DP) cells to promote hair growth. It was found that co-culture of Wntexpressing cells with DP cells maintains hair inductivity. In addition,it was found that agents, such as inhibitors of GSK3β kinase, e.g.,lithium chloride or similar small ions, which can mimic an effect of Wntpromoted signal transduction, e.g., inhibition of β-cateninphosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation ofβ-catenin, can regulate the ability of DP cells to promoter hair growth.

Accordingly, in one aspect, the invention features a method of promotinghair growth in a subject. The method includes inducing or mimicking theeffects of Wnt promoted signal transduction, e.g., by increasing thelevel of Wnt protein or administering an agent which mimics an effect ofWnt promoted signal transduction, e.g., inhibition of β-cateninphosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation ofβ-catenin, to thereby promote hair growth.

In a preferred embodiment, the Wnt protein is: Wnt3, e.g., Wnt3a or Wnt3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferredembodiment, the Wnt protein is Wnt3, most preferably Wnt3a.

In a preferred embodiment, Wnt is increased by administering an agentwhich increases the level of Wnt protein production and/or activity. Anagent which increases the level of Wnt protein and/or which mimics aneffect of Wnt promoted signal transduction can be one or more of: a Wntpolypeptide or a functional fragment or analog thereof, a nucleotidesequence encoding a Wnt polypeptide or functional fragment or analogthereof, an agent which increases Wnt nucleic acid expression, e.g., asmall molecule which binds to the promoter region of Wnt; an agent whichmimics an effect of Wnt promoted signal transduction, e.g., inhibitionof β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, oraccumulation of β-catenin. Examples of agents which can mimic an effectof Wnt promoted signal transduction include: an inhibitor of GSK3βkinase, e.g., lithium chloride or similar small ions; agents which bindFrizzled (Frz) (a cell surface receptor) and mimic Wnt binding, e.g.,anti-Frizzled antibodies, or other naturally or non-naturally occurringFrizzled binding ligands.

In a preferred embodiment, Wnt is increased by administering, e.g.,introducing, a nucleotide sequence encoding a Wnt polypeptide orfunctional fragment or analog thereof, into a particular cell, e.g., anepidermal cell or a DP cell, in the subject. The nucleotide sequence canbe a genomic sequence or a cDNA sequence. The nucleotide sequence caninclude: a Wnt coding region; a promoter sequence, e.g., a promotersequence from a Wnt gene or from another gene; an enhancer sequence,e.g., 5′ untranslated region (UTR), e.g., a 5′ UTR from a Wnt gene orfrom another gene, a 3′ UTR, e.g., a 3′ UTR from a Wnt gene or fromanother gene; a polyadenylation site; an insulator sequence.

In another preferred embodiment, the level of Wnt protein is increasedby increasing the level of expression of an endogenous Wnt gene, e.g.,by increasing transcription of the Wnt gene. In a preferred embodiment,transcription of the Wnt gene is increased by: altering the regulatorysequences of the endogenous Wnt gene, e.g., by the addition of apositive regulatory element (such as an enhancer or a DNA-binding sitefor a transcriptional activator); the deletion of a negative regulatoryelement (such as a DNA-binding site for a transcriptional repressor)and/or replacement of the endogenous regulatory sequence, or elementstherein, with that of another gene, thereby allowing the coding regionof the Wnt gene to be transcribed more efficiently.

In another embodiment, the method can include introducing a cell into asubject, e.g., a cell expressing Wnt. In a preferred embodiment, thecell expresses a Wnt protein, e.g., a Wnt 3, Wnt 4, or Wnt 7, or afragment or an analog thereof. In another preferred embodiment, the cellhas been genetically modified to cause the expression of Wnt, e.g., thecell has been genetically modified to express a Wnt protein, or afragment or an analog thereof, or the cell has been genetically modifiedto introduce a nucleic acid sequence, e.g., a regulatory sequence, e.g.,a promoter or an enhancer, that causes or increases the expression ofthe endogenous Wnt. In a preferred embodiment, the promoter of theendogenous Wnt gene has been replaced by another promoter, e.g., by apromoter from another gene. The cell can be an autologous, allogeneic,or xenogeneic cell, but is preferably autologous. The autologous cell ispreferably from a subject characterized with hair loss. The manipulatedcell can be any cell type, e.g., a fibroblast, a keratinocyte, anepithelial cell, an endothelial cell, a glial cell, a neural cell, alymphocyte, a bone marrow cell, and a muscle cell. Preferably the cellis an epithelial cell, e.g., an epidermal cell, a hair follicle cell, adermal papilla cell. The cell can be introduced into a subject toincrease Wnt activity.

In a preferred embodiment, the level of Wnt, e.g., Wnt 3, Wnt 4, or Wnt7, is increased over a sustained period of time, e.g., a period equal toor greater than 2, 10, 14, 30, 60, 90, or 180 days. E.g., a cellexpressing a Wnt protein, fragment, or analog can be supplied, e.g., byany method described herein, whereby Wnt is released over a sustainedperiod of time, e.g., a period equal to or greater than 2, 10, 14, 30,60, 90, or 180 days.

In a preferred embodiment, the agent which increases the level of Wntprotein or mimics an effect of Wnt promoted signal transduction isadministered, e.g., by topically administering the agent; systemicallyadministering the agent; orally administering the agent; or injectingthe agent, preferably dermally or subcutaneously. In preferredembodiments, the compound is administered using a suitable deliveryvehicle, for example, a surfactant or an agent which increasespermeability in the skin, e.g., an SDS or DMSO containing formulation.Preferably, the agent is included in a composition for topical use,e.g., the composition is a gel, cream, or liquid. In a preferredembodiment, the agent is administered: by continuous administration,e.g., the agent is administered with sufficient frequency such that theaffect on the Wnt protein level or Wnt promoted signal transduction ismaintained for a selected period, e.g., 10, 20, 30, 50, 90, 180, 365days or more. In another preferred embodiment, administration of theagent is repeated, e.g., is repeated at least 1, 2, 3, 5, 10, 20 or moretimes.

In a preferred embodiment, hair growth is promoted on: the subject'sscalp; the subject's face, e.g., beard and/or mustache facial hairgrowth is promoted.

In a preferred embodiment, the subject has an insufficient amount ofhair or an insufficient rate of hair growth. In a preferred embodiment,the subject suffers from genetic pattern baldness; suffers from ahormonal disorder which decreases hair growth; has received a treatment,e.g., radiation, or chemotherapy, or a drug which inhibits hair growth;or has had a surgical procedure, e.g., skin graft, which is in need ofhair growth.

In another aspect, the invention features a method of inhibiting hairgrowth in a subject. The method includes inhibiting the level of Wntprotein or inhibiting an effect of Wnt promoted signal transduction, inthe subject.

In a preferred embodiment, the Wnt protein is: Wnt3, e.g., Wnt3a or Wnt3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferredembodiment, the Wnt protein is Wnt3, most preferably Wnt3a.

In a preferred embodiment, Wnt is inhibited by administering an agentwhich inhibits Wnt protein production levels and/or is a Wnt antagonist.An agent which inhibits Wnt can be one or more of: a Frizzled protein orWnt binding portion thereof; a Wnt nucleic acid molecule which can bindto a cellular Wnt nucleic acid sequence, e.g., mRNA, and inhibitexpression of the protein, e.g., an antisense molecule or Wnt ribozyme;an antibody that specifically binds to Wnt protein, e.g., an antibodythat disrupts Wnt's ability to bind to its natural cellular target,e.g., disrupts Wnt's ability to bind to Frizzled; an antibody thatspecifically binds to Frizzled, e.g., an antibody that disruptsFrizzled's ability to bind to Wnt; a mutated inactive Wnt protein orfragment which binds to Frizzled but does not activate the Wnt signalingpathway; an agent which decreases Wnt gene expression, e.g., a smallmolecule which binds the promoter of Wnt.

In another preferred embodiment, Wnt is inhibited by decreasing thelevel of expression of an endogenous Wnt gene, e.g., by decreasingtranscription of the Wnt gene. In a preferred embodiment, transcriptionof the Wnt gene can be decreased by: altering the regulatory sequencesof the endogenous Wnt gene, e.g., by the addition of a negativeregulatory sequence (such as a DNA-binding site for a transcriptionalrepressor).

In a preferred embodiment, the agent which decreases the level of Wntprotein of inhibits an effect of Wnt promoted signal transduction isadministered, e.g., by topically administering the agent; systemicallyadministering the agent; orally administering the agent; or injectingthe agent, preferably dermally or subcutaneously. In preferredembodiments, the compound is administered using a suitable deliveryvehicle, for example, a surfactant or an agent which increasespermeability in the skin, e.g., an SDS or DMSO containing formulation.Preferably, the agent is included in a composition for topical use,e.g., the composition is a gel, cream, or liquid. In a preferredembodiment, the agent is administered: by continuous administration,e.g., the agent is administered with sufficient frequency such that theaffect on the Wnt protein level or Wnt promoted signal transduction ismaintained for a selected period, e.g., 10, 20, 30, 50, 90, 180, 365days or more. In another preferred embodiment, administration of theagent is repeated, e.g., is repeated at least 1, 2, 3, 5, 10, 20 or moretimes.

In a preferred embodiment, hair growth is inhibited on: the subject'sscalp; the subject's face, e.g., beard and/or mustache facial hairgrowth or eyebrow growth is inhibited; the subject's body hair growth isinhibited, e.g., hair growth is inhibited on the subject's back, legs,chest, armpits.

In another aspect, the invention features a method of promoting hairgrowth in a subject. The method includes activating or increasingactivation of the Wnt-β-catenin signaling pathway.

In a preferred embodiment, activation of the Wnt-β-catenin pathway isincreased by administering an agent which increases the level of Wntprotein production and/or which mimics an effect of Wnt promoted signaltransduction, e.g., inhibition of β-catenin phosphorylation, e.g., byinhibition of GSK3β kinase, or accumulation of β-catenin. An agent whichincreases the level of Wnt protein and/or which mimics an effect of Wntpromoted signal transduction can be one or more of: a Wnt polypeptide ora functional fragment or analog thereof as described herein; anucleotide sequence encoding a Wnt polypeptide or functional fragment oranalog thereof as described herein; an agent which increases Wnt nucleicacid expression, e.g., a small molecule which binds to the promoterregion of Wnt; an agent which mimics an effect of Wnt promoted signaltransduction, e.g., inhibition of β-catenin phosphorylation, e.g., byinhibition of GSK3β kinase, or accumulation of β-catenin. Examples ofagents which can mimic Wnt promoted signal transduction includeinhibitors of GSK3β, e.g., lithium chloride or similar small ions,agents which bind Frizzled and mimic Wnt binding, e.g., anti-Frizzledantibodies, or other naturally or non-naturally occurring Frizzledbinding ligands. In other embodiments, the level of Wnt protein can beincreased by increasing the level of expression of an endogenous Wntgene, e.g., by increasing transcription of the Wnt gene, as describedherein. In another preferred embodiment, activation of the Wnt-β-cateninpathway is increased by administering: an agent which increasesβ-catenin protein production or activity, e.g., an agent which decreasesphosphorylation of β-catenin and/or which increases β-cateninaccumulation, e.g., a β-catenin polypeptide or a fragment or analogthereof, a nucleic acid sequence encoding a β-catenin polypeptide orfragments or analogs thereof; an agent which increases LEF-1 proteinproduction and/or activity, e.g., an LEF-1 polypeptide or a fragment oranalog thereof, a nucleic acid sequence encoding an LEF-1 polypeptide orfragments or analogs thereof.

In another preferred embodiment, the method can include introducing acell, e.g., a cell which expresses and preferably secretes a proteininvolved in the activation of the Wnt-β-catenin signaling pathway, intoa subject. In a preferred embodiment, the cell has been geneticallymodified to express: a Wnt protein, or a fragment or an analog thereof;a β-catenin protein, or fragment or analog thereof; an LEF-1 protein, ora fragment or analog thereof. In a preferred embodiment, the cellexpresses a Wnt protein, e.g., a Wnt 3, Wnt 4, or Wnt 7, or a fragmentor an analog thereof. In another preferred embodiment, the cell has beengenetically modified to cause the expression of Wnt, e.g., the cell hasbeen genetically modified to express a Wnt protein, or a fragment or ananalog thereof, or the cell has been genetically modified to introduce anucleic acid sequence, e.g., a regulatory sequence, e.g., a promoter oran enhancer, that causes or increases the expression of the endogenousWnt. In a preferred embodiment, the promoter of the endogenous Wnt genehas been replaced by another promoter, e.g., by a promoter from anothergene. The cell can be an autologous, allogeneic, or xenogeneic cell, butis preferably autologous. The autologous cell is preferably from asubject characterized with hair loss. The manipulated cell can be anycell type, e.g., a fibroblast, a keratinocyte, an epithelial cell.Preferably the cell is an epithelial cell, e.g., an epidermal cell, ahair follicle cell, a dermal papilla cell. The cell can be introducedinto a subject to increase Wnt activity.

In a preferred embodiment, the level of Wnt, e.g., Wnt 3, Wnt 4, or Wnt7, is increased over a sustained period of time, e.g., a period equal toor greater than 2, 10, 14, 30, 60, 90, or 180 days. E.g., a cellexpressing a Wnt protein, fragment, or analog can be supplied, e.g., byany method described herein, whereby Wnt is released over a sustainedperiod of time, e.g., a period equal to or greater than 2, 10, 14, 30,60, 90, or 180 days. The cell can be introduced into a subject, e.g., toincrease the level of the protein involved in activation of theWnt-β-catenin signaling pathway.

In a preferred embodiment, the agent is administered, e.g., by topicallyadministering the agent; systemically administering the agent; orallyadministering the agent; or injecting the agent, preferably dermally orsubcutaneously. In preferred embodiments, the compound is administeredusing a suitable delivery vehicle, for example, a surfactant or an agentwhich increases permeability in the skin, e.g., an SDS or DMSOcontaining formulation. Preferably, the agent is included in acomposition for topical use, e.g., the composition is a gel, cream, orliquid. In a preferred embodiment, the agent is administered: bycontinuous administration, e.g., the agent is administered withsufficient frequency such that the affect on the Wnt-β-catenin signalingpathway is maintained for a selected period, e.g., 10, 20, 30, 50, 90,180, 365 days or more. In another preferred embodiment, administrationof the agent is repeated, e.g., is repeated at least 1, 2, 3, 5, 10, 20or more times.

In a preferred embodiment, hair growth is promoted on: the subject'sscalp; the subject's face, e.g., beard and/or mustache facial hairgrowth is promoted.

In a preferred embodiment, the subject has an insufficient amount ofhair or an insufficient rate of hair growth. In a preferred embodiment,the subject suffers from genetic pattern baldness; suffers from ahormonal disorder which decreases hair growth; has received a treatment,e.g., radiation, or chemotherapy, or a drug which inhibits hair growth;or has had a surgical procedure, e.g., skin graft, which is in need ofhair growth.

In another aspect, the invention features a method of inhibiting hairgrowth in a subject. The method includes inhibiting activation of theWnt-β-catenin signaling pathway.

In a preferred embodiment, activation of the Wnt-β-catenin pathway isinhibited by administering an agent which decreases the level of Wntprotein production and/or decreases an effect of Wnt promoted signaltransduction. An agent which inhibits the level of Wnt protein or is aWnt antagonist can be one or more of: a Frizzled protein or Wnt bindingportion thereof; a Wnt nucleic acid molecule which can bind to acellular Wnt nucleic acid sequence, e.g., mRNA, and inhibit expressionof the protein, e.g., an antisense molecule or Wnt ribozyme; an antibodythat specifically binds to Wnt protein, e.g., an antibody that disruptsWnt's ability to bind to its natural cellular target, e.g., disruptsWnt's ability to bind to a Frizzledreceptor protein; an antibody thatspecifically binds to Frizzled, e.g., an antibody that disrupts aFrizzled's ability to bind to Wnt; a mutated inactive Wnt protein orfragment which binds to Frizzled but does not activate the Wnt signalingpathway; an agent which decreases Wnt gene expression, e.g., a smallmolecule which binds the promoter of Wnt; an agent which decreases anactivity of Wnt, e.g., an agent which increases phosphorylation ofβ-catenin. In other embodiments, the level of Wnt protein can beinhibited by decreasing the level of expression of an endogenous Wntgene, e.g., by decreasing transcription of the Wnt gene, as describedherein. In another preferred embodiment, activation of the Wnt-β-cateninpathway is inhibited by administering: an agent which inhibits β-cateninprotein production or inhibits an effect of Wnt promoted signaltransduction, e.g., an agent which increases phosphorylation ofβ-catenin and/or which decreases β-catenin accumulation; an agent whichinhibits LEF-1 protein production and/or activity.

In a preferred embodiment, the agent is administered, e.g., by topicallyadministering the agent; systemically administering the agent; orallyadministering the agent; or injecting the agent, preferably dermally orsubcutaneously. In preferred embodiments, the compound is administeredusing a suitable delivery vehicle, for example, a surfactant or an agentwhich increases permeability in the skin, e.g., an SDS or DMSOcontaining formulation. Preferably, the agent is included in acomposition for topical use, e.g., the composition is a gel, cream, orliquid. In a preferred embodiment, the agent is administered: bycontinuous administration, e.g., the agent is administered withsufficient frequency such that the effect on the Wnt-β-catenin signalingpathway is maintained for a selected period, e.g., 10, 20, 30, 50, 90,180, 365 days or more. In another preferred embodiment, administrationof the agent is repeated, e.g., is repeated at least 1, 2, 3, 5, 10, 20or more times.

In a preferred embodiment, hair growth is inhibited on: the subject'sscalp; the subject's face, e.g., beard and/or mustache facial hairgrowth or eyebrow growth is inhibited; the subject's body hair growth isinhibited, e.g., hair growth is inhibited on the subject's back, legs,chest, armpits.

In another aspect, the invention features a method of evaluating thestatus of hair growth/hair loss in a subject. The method includesevaluating, e.g., detecting, the presence or absence of a genetic lesionin a Wnt gene, or evaluating, e.g., detecting, misexpression of the Wntgene.

In one embodiment, the method includes evaluating whether a subject isat risk for hair loss. The method includes evaluating, e.g., detecting,a genetic lesion in a Wnt gene, or evaluating, e.g., detecting,underexpression of the Wnt gene, to thereby determine if a subject is atrisk for hair loss.

In a preferred embodiment, the Wnt gene or protein is: Wnt3, e.g., Wnt3aor Wnt 3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferredembodiment, the Wnt protein is Wnt3, most preferably Wnt3a.

In preferred embodiment, the method includes evaluating in a sample ofcells from the subject for the presence or absence of a genetic lesion,e.g., a mutation in the gene encoding a Wnt protein. The presence of agenetic lesion is indicative of a risk of hair loss in a subject. Thecell sample can be of any cell type, e.g., a fibroblast, a keratinocyte,an epithelial cell, an endothelial cell, a glial cell, a neural cell, alymphocyte, a bone marrow cell, and a muscle cell.

In another preferred embodiment, the method includes evaluating in asample of cells, e.g., a sample of epidermal cells from the hairfollicle of a subject, for the expression levels of the Wnt to determineunderexpression. Underexpression of Wnt is indicative of a risk of hairloss.

In a preferred embodiment, the genetic lesions is evaluated bycontacting the sample with a nucleic acid probe capable of hybridizingto Wnt mRNA, e.g., a labeled probe. In another preferred embodiment,expression of Wnt is evaluated with an antibody capable of binding toWnt protein, e.g., a labeled antibody.

In another embodiment, the method includes evaluating hair growth in asubject. The method includes evaluating, e.g., detecting, absence orpresence of a genetic lesion in a Wnt gene, or evaluating, e.g.,detecting, overexpression of the Wnt gene, to thereby evaluate whetherhair growth is likely in a subject.

In a preferred embodiment, the Wnt gene or protein is: Wnt3, e.g., Wnt3aor Wnt 3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferredembodiment, the Wnt protein is Wnt3, most preferably Wnt3a.

In a preferred embodiment, the method includes evaluating in a sample ofcells from the subject for the presence or absence of a genetic lesion,e.g., a mutation in the gene encoding a Wnt protein. The absence of agenetic lesion is indicative of a potential for hair growth. The cellsample can be of any cell type, e.g., a fibroblast, a keratinocyte, anepithelial cell, an endothelial cell, a glial cell, a neural cell, alymphocyte, a bone marrow cell, and a muscle cell.

In another preferred embodiment, the method includes evaluating in asample of cells, e.g., a sample of epidermal cells from the hairfollicle of a subject, for the expression levels of Wnt to determineoverexpression. Overexpression of Wnt is indicative of a potential forhair growth.

In a preferred embodiment, the genetic lesions is evaluated bycontacting the sample with a nucleic acid probe capable of hybridizingto Wnt mRNA, e.g., a labeled probe. In another preferred embodiment,expression of Wnt is evaluated with an antibody capable of binding toWnt protein, e.g., a labeled antibody.

In another aspect, the invention features a method of evaluating theability of an epidermal cell to promote hair growth or hair loss in asubject. The method includes evaluating, e.g., detecting, the presenceor absence of a genetic lesion in a Wnt gene, or evaluating, e.g.,detecting, misexpression of the Wnt gene.

In a preferred embodiment, the ability of an epidermal cell to promotehair growth or hair loss is evaluated in vitro.

In one embodiment, the method includes evaluating the ability of anepidermal cell to promote hair loss. The method includes evaluating,e.g., detecting, a genetic lesion in a Wnt gene, or evaluating, e.g.,detecting, underexpression of the Wnt gene.

In a preferred embodiment, the Wnt gene or protein is: Wnt3, e.g., Wnt3aor Wnt 3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferredembodiment, the Wnt protein is Wnt 3, most preferably, Wnt3a.

In preferred embodiment, the method includes evaluating in a sample ofepidermal cells from the subject for the presence or absence of agenetic lesion, e.g., a mutation in the gene encoding a Wnt protein. Thepresence of a genetic lesion is indicative of a risk of hair loss in asubject.

In another preferred embodiment, the method includes evaluating in asample of epidermal cells, for the expression levels of the Wnt todetermine underexpression. Underexpression of Wnt is indicative of arisk of hair loss.

In a preferred embodiment, the genetic lesions is evaluated bycontacting the sample with a nucleic acid probe capable of hybridizingto Wnt mRNA, e.g., a labeled probe. In another preferred embodiment,expression of Wnt is evaluated with an antibody capable of binding toWnt protein, e.g., a labeled antibody.

In another embodiment, the method includes evaluating the ability of anepidermal cell to promote hair growth. The method includes evaluating,e.g., detecting, absence or presence of a genetic lesion in a Wnt gene,or evaluating, e.g., detecting, overexpression of the Wnt gene.

In a preferred embodiment, the Wnt gene or protein is: Wnt3, e.g., Wnt3aor Wnt 3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferredembodiment, the Wnt protein is Wnt3, most preferably, Wnt3a.

In a preferred embodiment, the method includes evaluating in a sample ofepidermal cells from the subject for the presence or absence of agenetic lesion, e.g., a mutation in the gene encoding a Wnt protein. Theabsence of a genetic lesion is indicative of a potential for hairgrowth.

In another preferred embodiment, the method includes evaluating in asample of epidermal cells for the expression levels of Wnt to determineoverexpression. Overexpression of Wnt is indicative of a potential forhair growth.

In a preferred embodiment, the genetic lesions is evaluated bycontacting the sample with a nucleic acid probe capable of hybridizingto Wnt mRNA, e.g., a labeled probe. In another preferred embodiment,expression of Wnt is evaluated with an antibody capable of binding toWnt protein, e.g., a labeled antibody.

In another aspect, the invention features a method for identifying acompound capable of promoting hair growth. The method includes:contacting a cell capable of expressing a Wnt polypeptide with a testcompound; and determining the level of Wnt polypeptide or nucleic acidexpression, wherein a compound capable of increasing Wnt polypeptide ornucleic acid expression is indicative of a compound capable of promotinghair growth.

In a preferred embodiment, the Wnt protein is: Wnt3, e.g., Wnt3a or Wnt3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferredembodiment, the Wnt protein is Wnt3, most preferably Wnt3a.

In a preferred embodiment, the compound is a Wnt fragment or analog.

In a preferred embodiment, the method further includes evaluating acontrol cell, e.g., an identical cell which is not treated with thecompound.

In a preferred embodiment, the cell is: an epidermal cell, e.g., anepidermal cell from a hair follicle; a DP cell.

In a preferred embodiment, Wnt nucleic acid expression is evaluatedusing a nucleic acid probe, e.g., a labeled probe, capable ofhybridizing to a Wnt nucleic acid molecule, e.g., Wnt mRNA. In apreferred embodiment, Wnt nucleic acid expression, e.g., DNA expression,is evaluated by contacting a compound with a Wnt nucleic acid molecule,e.g., a regulatory sequence of a Wnt nucleic acid molecule, andevaluating Wnt transcription, in vitro or in vivo, e.g., Wnttranscription is evaluated by determining a cell activity, e.g., using amarker gene, e.g., a lacZ gene or green fluorescence protein (GFP) gene,fused to the regulatory sequence of Wnt and following production of themarker.

In a preferred embodiment, Wnt polypeptide expression is evaluated usingan anti-Wnt antibody, e.g., a labeled anti-Wnt antibody.

In another aspect, the invention features a method for identifying acompound capable of inhibiting hair growth. The method includes:contacting a cell capable of expressing a Wnt polypeptide with a testcompound; and determining the level of Wnt polypeptide or nucleic acidexpression in the presence and absence of the compound, wherein acompound capable of decreasing Wnt polypeptide or nucleic acidexpression is indicative of a compound capable of inhibiting hairgrowth.

In a preferred embodiment, the Wnt polypeptide is: Wnt3, e.g., Wnt3a orWnt 3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferredembodiment, the Wnt protein is Wnt3, most preferably Wnt3a.

In a preferred embodiment, Wnt nucleic acid expression is evaluatedusing a nucleic acid probe, e.g., a labeled probe, capable ofhybridizing to a Wnt nucleic acid molecule, e.g., Wnt mRNA. In preferredembodiment, Wnt nucleic acid expression, e.g., DNA expression, isevaluated by contacting a compound with a Wnt nucleic acid molecule,e.g., a regulatory sequence of a Wnt nucleic acid molecule, andevaluating Wnt transcription, in vitro or in vivo, Wnt transcription isevaluated by determining a cell activity, e.g., using a marker gene,e.g., a lacZ gene or a GFP gene, fused to the regulatory sequence of Wntand following production of the marker.

In a preferred embodiment, Wnt polypeptide expression is evaluated usingan anti-Wnt antibody, e.g., a labeled anti-Wnt antibody.

In another aspect, the invention features a method of culturing a DPcell. For example, a human or non-human, e.g., rodent, e.g., rat ormouse, DP cell. The method includes culturing the DP cell in thepresence of an increased level of Wnt, another protein involved inactivating the Wnt-β-catenin signaling pathway, e.g., β-catenin and/orLEF-1, and/or an agent which mimics an effect of Wnt promoted signaltransduction, e.g., inhibition of β-catenin phosphorylation, e.g., byinhibition of GSK3β kinase, or accumulation of β-catenin.

In a preferred embodiment, the level of Wnt is increased over DP cellsin the absence of Wnt.

In a preferred embodiment, the Wnt protein is: Wnt3, e.g., Wnt3a or Wnt3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferredembodiment, the Wnt protein is Wnt3, most preferably Wnt3a.

In a preferred embodiment, the DP cell is propagated in vitro. In apreferred embodiment, the DP cell is cultured to increase the number ofDP cells.

In a preferred embodiment, a Wnt polypeptide or a functional fragment oranalog thereof is added to the culture. In another preferred embodiment,an agent which mimics an effect of Wnt promoted signal transduction,e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition ofGSK3β kinase, or accumulation of β-catenin, is added to the culture.Examples of agents which can mimic an effect of Wnt promoted signaltransduction include inhibitors of GSK3β kinase such as lithium chlorideor similar small ions.

In another preferred embodiment, the DP cell is cultured in the presenceof a cell which expresses a Wnt polypeptide or a functional fragment oranalog thereof.

In a preferred embodiment, the DP cell is obtained from a subject,cultured with an increased level of Wnt, or an agent which mimics aneffect of Wnt promoted signal transduction, e.g., inhibition ofβ-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, oraccumulation of β-catenin, and then returned to the same or a differentsubject.

In a preferred embodiment, the DP cell is maintained in culture and thenthe cultured DP cells are returned to the same or a different subject toincrease the amount of hair growth in the individual.

In another preferred embodiment, the invention features a method ofproviding and maintaining a dermal papilla cell graft, e.g., a DP graftfor hair transplantation procedures. The method includes culturing a DPcell or DP cells in the presence of Wnt or a fragment or analog thereof,another protein involved in activating the Wnt-β-catenin signalingpathway (e.g., β-catenin and/or LEF-1) or a fragment or analog thereof,and/or an agent which mimics an effect of Wnt promoted signaltransduction, e.g., inhibition of β-catenin phosphorylation, e.g., byinhibition of GSK3β kinase, or accumulation of β-catenin.

In a preferred embodiment, the DP cell is propagated in vitro. In apreferred embodiment, the DP cell is propagated in vitro to increase thenumber of DP cells.

In a preferred embodiment, a Wnt polypeptide or a functional fragment oranalog thereof is added to the culture. In another preferred embodiment,an agent which mimics an effect of Wnt promoted signal transduction,e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition ofGSK3β kinase, or accumulation of β-catenin, is added to the culture.Examples of agents which mimic an effect of Wnt promoted signaltransduction include inhibitors of GSK3β kinase such as lithium chlorideor similar small ions.

In another preferred embodiment, the DP cell is cultured in the presenceof a cell which expresses a Wnt polypeptide or a functional fragment oranalog thereof.

In a preferred embodiment, the DP cell is obtained from a subject,cultured with an increased level of Wnt, or an agent which mimics aneffect of Wnt promoted signal transduction, e.g., inhibition ofβ-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, oraccumulation of β-catenin, and then returned to the same or a differentsubject.

In another aspect, the invention features a media for culturing DP cellswhich includes a Wnt polypeptide or a functional fragment or analogthereof, or an agent which mimics an effect of Wnt promoted signaltransduction, e.g., inhibition of β-catenin phosphorylation, e.g., byinhibition of GSK3β kinase, or accumulation of β-catenin. Examples ofagents which mimic an effect of Wnt promoted signal transduction includeinhibitors of GSK3β kinase such as lithium chloride or similar smallions.

In another aspect, the invention features a method of promoting ormaintaining anagen phase gene expression of DP cells. The methodincludes increasing the level of Wnt protein or mimicking an effect ofWnt promoted signal transduction, e.g., inhibition of β-cateninphosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation ofβ-catenin, to thereby promote or maintain anagen phase gene expressionin the DP cells. In another preferred embodiment, the method includesincreasing activation of the Wnt-β-catenin signaling pathway, to therebypromote or maintain anagen phase gene expression in DP cells.

In a preferred embodiment, the Wnt protein is: Wnt3, e.g., Wnt3a or Wnt3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferredembodiment, the Wnt protein is Wnt3, most preferably Wnt3a.

In a preferred embodiment, Wnt level is increased or an effect of Wntpromoted signal transduction is mimicked by administering an agent whichincreases the level of Wnt protein production and/or which mimics aneffect of Wnt promoted signal transduction, e.g., inhibition ofβ-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, oraccumulation of β-catenin. An agent which increases the level of Wntprotein and/or mimics an effect of Wnt promoted signal transduction canbe one or more of: a Wnt polypeptide or a functional fragment or analogthereof, as described herein; a nucleotide sequence encoding a Wntpolypeptide or functional fragment or analog thereof, as describedherein; an agent which increases Wnt nucleic acid expression, e.g., asmall molecule which binds to the promoter region of Wnt; an agent whichmimics an effect of Wnt promoted signal transduction, e.g., inhibitionof β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, oraccumulation of β-catenin. Examples of agents which can mimic Wntpromoted signal transduction include inhibitors of GSK3β, e.g., lithiumchloride or similar small ions, agents which bind Frizzled and mimic Wntbinding, e.g., anti-Frizzled antibodies, or other naturally ornon-naturally occurring Frizzled binding ligands.

In a preferred embodiment, the method can be performed in vitro or invivo. For example, the DP cells can be maintained in anagen phase inculture, and then administered to a subject, e.g., to increase hairgrowth. Such methods can include maintaining DP cells in culture in thepresence of Wnt or an agent which mimics an effect of Wnt promotedsignal transduction, e.g., inhibition of β-catenin phosphorylation,e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin. Inone embodiment, Wnt and/or an agent which mimics an effect of Wntpromoted signal transduction, e.g., an inhibitor of GSK3β kinase, e.g.,lithium chloride or similar small ions, can be added to the culture. Inanother embodiment, the DP cell can be co-cultured with a cell whichexpresses Wnt, e.g., a cell which naturally expressed Wnt or has beengenetically engineered to express Wnt. DP cells maintained in anagenphase can then be used, e.g., in DP graft procedures. The DP cells canbe obtained from the subject who will be receiving the DP graft (i.e.,autologous cells), or can be obtained from a different subject (e.g.,allogeneic or xenogeneic cells).

In a preferred embodiment, Wnt is increased by administering, e.g.,introducing, a nucleotide sequence encoding a Wnt polypeptide orfunctional fragment or analog thereof, into a particular cell, e.g., anepidermal cell or a DP cell, and/or into a subject. The nucleotidesequence can be a genomic sequence or a cDNA sequence. The nucleotidesequence can include: a Wnt coding region; a promoter sequence, e.g., apromoter sequence from a Wnt gene or from another gene; an enhancersequence, e.g., 5′ untranslated region (UTR), e.g., a 5′ UTR from a Wntgene or from another gene, a 3′ UTR, e.g., a 3′ UTR from a Wnt gene orfrom another gene; a polyadenylation site; an insulator sequence.

In another preferred embodiment, the level of Wnt protein is increasedby increasing the level of expression of an endogenous Wnt gene, e.g.,by increasing transcription of the Wnt gene. In a preferred embodiment,transcription of the Wnt gene is increased by: altering the regulatorysequences of the endogenous Wnt gene, e.g., by the addition of apositive regulatory element (such as an enhancer or a DNA-binding sitefor a transcriptional activator); the deletion of a negative regulatoryelement (such as a DNA-binding site for a transcriptional repressor)and/or replacement of the endogenous regulatory sequence, or elementstherein, with that of another gene, thereby allowing the coding regionof the Wnt gene to be transcribed more efficiently.

In another preferred embodiment, the method can include introducing acell, e.g., a cell which expresses and preferably secretes a Wntprotein, into a subject. In a preferred embodiment, the cell has beengenetically modified to express a Wnt protein, or a fragment or ananalog thereof The cell can be an autologous, allogeneic, or xenogeneiccell, but is preferably autologous. The cell can be any cell type, e.g.,a fibroblast, a keratinocyte, an epithelial cell, an endothelial cell.Preferably the cell is an epithelial cell, e.g., an epidermal cell or aDP cell. The cell can be introduced into a subject to increase the levelof Wnt protein.

In a preferred embodiment, the agent which increases the level of Wntprotein and/or mimics an effect of Wnt promoted signal transduction isadministered, e.g., by topically administering the agent; systemicallyadministering the agent; orally administering the agent; or injectingthe agent, preferably dermally or subcutaneously. In preferredembodiments, the compound is administered using a suitable deliveryvehicle, for example, a surfactant or an agent which increasespermeability in the skin, e.g., an SDS or DMSO containing formulation.Preferably, the agent is included in a composition for topical use,e.g., the composition is a gel, cream, or liquid. In a preferredembodiment, the agent is administered: by continuous administration,e.g., the agent is administered with sufficient frequency such that theaffect on the Wnt protein level is maintained for a selected period,e.g., 10, 20, 30, 50, 90, 180, 365 days or more. In another preferredembodiment, administration of the agent is repeated, e.g., is repeatedat least 1, 2, 3, 5, 10, 20 or more times.

In a preferred embodiment, anagen phase gene expression is promoted ormaintained in: the subject's scalp; the subject's face, e.g., upper lipand/or chin.

In another aspect, the invention features a method of promoting ormaintaining hair inductive activity. The method includes increasing thelevel of Wnt protein and/or mimicking an effect of Wnt promoted signaltransduction, e.g., inhibition of β-catenin phosphorylation, e.g., byinhibition of GSK3β kinase, or accumulation of β-catenin, to therebypromote or maintain hair inductive activity.

In a preferred embodiment, the Wnt protein is: Wnt3, e.g., Wnt3a or Wnt3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferredembodiment, the Wnt protein is Wnt3, most preferably Wnt3a.

In a preferred embodiment, Wnt is increased by administering an agentwhich increases the level of Wnt protein production and/or activity. Anagent which increases the level of Wnt protein can be one or more of: aWnt polypeptide or a functional fragment or analog thereof; a nucleotidesequence encoding a Wnt polypeptide or functional fragment or analogthereof; an agent which increases Wnt nucleic acid expression, e.g., asmall molecule which binds to the promoter region of Wnt; an agent whichmimics an effect of Wnt promoted signal transduction, e.g., inhibitionof β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, oraccumulation of β-catenin. Examples of agents which mimic an effect ofWnt promoted signal transduction include inhibitors of GSK3β kinase suchas lithium chloride or similar small ions.

In a preferred embodiment, Wnt is increased by administering, e.g.,introducing, a nucleotide sequence encoding a Wnt polypeptide orfunctional fragment or analog thereof, into a particular cell, e.g., anepidermal cell or a DP cell, in the subject. The nucleotide sequence canbe a genomic sequence or a cDNA sequence. The nucleotide sequence caninclude: a Wnt coding region; a promoter sequence, e.g., a promotersequence from a Wnt gene or from another gene; an enhancer sequence,e.g., 5′ untranslated region (UTR), e.g., a 5′ UTR from a Wnt gene orfrom another gene, a 3′ UTR, e.g., a 3′ UTR from a Wnt gene or fromanother gene; a polyadenylation site; an insulator sequence.

In another preferred embodiment, the level of Wnt protein is increasedby increasing the level of expression of an endogenous Wnt gene, e.g.,by increasing transcription of the Wnt gene. In a preferred embodiment,transcription of the Wnt gene is increased by: altering the regulatorysequences of the endogenous Wnt gene, e.g., by the addition of apositive regulatory element (such as an enhancer or a DNA-binding sitefor a transcriptional activator); the deletion of a negative regulatoryelement (such as a DNA-binding site for a transcriptional repressor)and/or replacement of the endogenous regulatory sequence, or elementstherein, with that of another gene, thereby allowing the coding regionof the Wnt gene to be transcribed more efficiently.

In another preferred embodiment, the method can include introducing acell, e.g., a cell which expresses and preferably secretes a Wntprotein, into a subject. In a preferred embodiment, the cell has beengenetically modified to express a Wnt protein, or a fragment or ananalog thereof. The cell can be an autologous, allogeneic, or xenogeneiccell, but is preferably autologous. The cell can be any cell type, e.g.,a fibroblast, a keratinocyte, an epithelial cell, an endothelial cell.Preferably the cell is an epithelial cell, e.g., an epidermal cell or aDP cell. The cell can be introduced into a subject to increase the levelof Wnt protein and/or to mimic an effect of Wnt promoted signaltransduction.

In a preferred embodiment, the agent which increases the level of Wntprotein and/or mimics Wnt promoted signal transduction is administered,e.g., by topically administering the agent; systemically administeringthe agent; orally administering the agent; or injecting the agent,preferably dermally or subcutaneously. In preferred embodiments, thecompound is administered using a suitable delivery vehicle, for example,a surfactant or an agent which increases permeability in the skin, e.g.,an SDS or DMSO containing formulation. Preferably, the agent is includedin a composition for topical use, e.g., the composition is a gel, cream,or liquid. In a preferred embodiment, the agent is administered: bycontinuous administration, e.g., the agent is administered withsufficient frequency such that the affect on the Wnt protein leveland/or the Wnt signaling pathway is maintained for a selected period,e.g., 10, 20, 30, 50, 90, 180, 365 days or more. In another preferredembodiment, administration of the agent is repeated, e.g., is repeatedat least 1, 2, 3, 5, 10, 20 or more times.

In a preferred embodiment, hair inductive activity is promoted ormaintained on: the subject's scalp; the subject's face, e.g., upperlipand/or chin.

A “treatment”, as used herein, includes any therapeutic treatment, e.g.,the administration of a therapeutic agent or substance, e.g., a drug.

The term “increasing hair growth” as used herein refers to increasingthe number or density or distribution of follicles or hair shafts orotherwise increasing the growth of hair.

As used herein, the term “subject” refers an animal, e.g., a mammal,e.g., a human. The mammal can be a human or non-human mammal, e.g., aswine, a bird, a cat, a dog, a monkey, a goat, or a rodent, e.g., a rator a mouse. The animal can be a transgenic animal, e.g., a transgenicrodent, e.g., a transgenic rat or mouse.

“Regulatory sequence” refers to any or all of the DNA sequences thatcontrols gene expression. An example of a regulatory sequence includes:a promoter, a positive regulatory element (such as an enhancer or aDNA-binding site for a transcriptional activator); a negative regulatoryelement (such as a DNA-binding site for a transcriptional repressor) andan insulator.

“Heterologous” refers to DNA or tissue which is derived from a differentspecies.

“Heterologous regulatory sequence” refers to a sequence which is not thenormal regulatory sequence of that gene

The terms “peptides”, “proteins”, and “polypeptides” are usedinterchangeably herein.

The term “small molecule”, as used herein, includes peptides,peptidomimetics, or non-peptidic compounds, such as organic molecules,having a molecular weight less than 2,000, preferably less than 1,000.

The term “effects of Wnt-promoted signal transduction” refers to one ormore of the biochemical effects (e.g., modulation of e.g., proteinbinding interactions, phosphorylation or transcription) in a cell, e.g.,a DP cell, initiated by Wnt signaling, e.g., by Wnt binding to Frizzled.Effects of Wnt promoted signal transduction can include Wnt binding toFrizzled; inhibition of GSK3β mediated phosphorylation; inhibition ofphosphorylation-dependent degradation of β-catenin; accumulation ofβ-catenin protein in the cytoplasm; stabilization of cellular β-catenin;β-catenin accumulation in the cytoplasm; β-catenin binding to Lef1;translocation of the β-catenin-Lef1 complex to the nucleus; andstimulation of transcription from associated genes. Components ofWnt-promoted signal transduction can include Frizzled protein, e.g.,Frizzled-7 (frz-7), disheveled proteins, e.g., disheveled-2 (dsh-2),GSK3, beta catenin, Lef1, and Lef/TFC. Other effects and components ofthe Wnt signaling pathway are described in Arias et al. (1999) Curr.Opin. Genet. & Dev. 9:447-454, which is incorporated herein byreference.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery that Wntproteins expressed in the follicular epithelium maintain anagen phasegene expression in dermal papilla cells and that hair inductive activityis also maintained by Wnt signaling.

Both formation of the hair follicle during embryonic development and thecyclical growth, quiescence and regeneration of the hair shaft duringthe hair cycle are dependent on reciprocal signaling between theepidermal and dermal components of the follicle.

In the Wnt signaling pathway, Wnt, which is a soluble molecule, bindsFrizzled (Frz), a cell surface receptor, found on various types ofcells. In the presence of disshelved, binding of Wnt to Frz results inthe inhibition of GSK3β mediated phosphorylation and subsequentphosphorylation-dependent degradation of β-catenin. Thus, Wnt bindingstabilizes cellular β-catenin. In the presence of Wnt binding, β-cateninaccumulates in the cytoplasm and binds to Lef1. The β-catenin-Lef1complex then translocates to the nucleus, where it mediatestranscriptional activation.

Production of a Transgenic Mouse Expressing Green Fluorescent Protein inDP Cells

A transgenic mouse line that specifically expresses green fluorescentprotein (GFP) in DP cells during the anagen (growth) phase of the haircycle was used to purify GFP expressing DP cells and study the signalsrequired to maintain GFP expression. The transgenic mouse line wasgenerated as described in Kishimoto et al. (1999) Proc. Natl Acad. SciUSA 96:7336-7341. It was found that isolated transgenic anagen hairfollicles show GFP fluorescence in the DP. The versican-GFP transgene isactive during anagen but is shut off during catagen and telogen of hairgrowth. Therefore, transgene expression was correlated with the presumedprofile of inductive activity in the DP.

RT-PCR Analysis

Total RNA was isolated and RT PCR performed as described in Kishimoto etal. (1999) Proc. Natl Acad. Sci. USA 96:7336-7341, using an annealingtemperature of 58° C. The primers employed are listed below. With theexception of Wnts 4, 5 and 7, the primers were designed to distinguishbetween mouse and chicken orthologues and the lack of cross reaction tochick sequences was confirmed on cDNA from feeder cells alone.

Wnt3: G CGC CCT GGC TCA CTA C (SEQ ID NO:1) and ATG CTG CTG CTG CTG GCC(SEQ ID NO:2) for 30 cycles Wnt4: TGA TCC AGA GGC AGG TGC AG (SEQ IDNO:3) and CTT CTC CAG TTC TCC ACT GC (SEQ ID NO:4) for 36 cycles Wnt5a:CTG TTG AC TGC ACC AGC TT (SEQ ID NO:5) and TCA AGG AAT GCC AGT ACC AGTACC AG (SEQ ID NO:6) for 30 cycles Frizzled-7: CTG CTA GAG GAC CGT GCC(SEQ ID NO:7) and AGG TGC GTT CCC AGT GCT (SEQ ID NO:8) for 36cyclesDisheveled-2: CAT CCT TCA GCA GTG TCA (SEQ ID NO:9) and CGT CAT TGT CATTCA GAG (SEQ ID NO:10) for 36 cycles GSK-3: CAG GGC ACC AGA GTT GAT (SEQID NO:11) and GCA GAA GCG GCG TTA TTG (SEQ ID NO:12) for 30 cycles-catenin: CCA CCA GCT AGG CGC ACT (SEQ ID NO:13) and GGG CTC AGA GGG TCCGAG (SEQ ID NO:14) for 30 cycles LEF-1: ACT GTC AGG CGA CAC TTC C (SEQID NO:15) and TGC ACG TTG GGA AGG AGC (SEQ ID NO:16) for 36 cycles Shh:for 30 cycles (SEQ ID NO:17 & 18) Patched-1: AGC CTC ACA GTA ACA CCC(SEQ ID NO:20) and TGT TCT CCT CCA GCA TGA (SEQ ID NO:21) for 36 cyclesGli-1: TTG GGG ATG CTG GAT GGG (SEQ ID NO:22) and CGG TCA CTG GCA TTGCTA (SEQ ID NO:23) for 36 cycles -actin: 5′-CCA CAC CCG CCA CCA GTTC-3′ (SEQ ID NO:24) and 5′-GAGGAAGAGGATGCGGCA-3′ (SEQ ID NO:25) for 26cycles GFP: 5′-TGCAGTGCTTCAGCCGCTAC-3′ (SEQ ID NO:26) AND5′-CTCGTTGGGGTCTTTGCTCA-3′ (SEQ ID NO:27) for 26 cycles.Cell Culture

Fresh mouse pelage DP cells were obtained from versican GFP transgenicnewborn skin by high-speed cell sorting (MoFLO) as described previouslyin Kishimoto et al., supra. Chick embryo fibroblasts (CEFs) wereinfected with RCASBP(A) retroviral vectors encoding chick Shh, asdescribed in Riddle et al. (1993) Cell 75:1401-1416, Wnt3a, as describedin Kengaku et al. (1998) Science 280:1274-1277, Wnt4, Wnt5a (Noramly andMorgan, in prep), or Wnt7a, as described in Riddle et al. (1995) Cell83:631-640, or vector alone as described, as described in Morgan et al.in Methods in Cell Biology (ed. Bonner-Fraser) pp.185-218 (AcademicPress, San Diego, 1996). GFP positive mouse DP cells were added onto30-50% confluent feeder cells to achieve a ratio of 1:3 (mouse:chick).For the flow cytometric analysis, cells were co-cultured in 24 welldishes for 48-96 hrs. For the grafting experiment co-cultured cells werepassaged 2-3 times in 10 cm dishes to generate the 2×10⁶ cells requiredfor each graft.

Flow Cytometry

Cells were trypsinized and resuspended in 0.5 ml PBS with 1% BSA. Flowcytometric analysis was performed by FACScan (Becton Dickinson). DPcells were labeled with mouse MHC class II monoclonal antibody andincubated with secondary IgG conjugated to PE. The PE negative aviancells fell below the lower gate in FIG. 3 and were excluded from theanalysis of GFP expression.

Reconstitution Assay

The reconstitution assay was performed as described in Kishimoto et al.,supra. Primary keratinocytes were prepared from 2 newborn pups per graftand combined with 2×10⁶ DP cells in the graft chamber. Hair growth wasmonitored two weeks after grafting and weekly thereafter.

Production of Cell Culture of GFP Expressing DP Cells

Anagen dermal papilla cells from the skin of the transgenic mousedescribed above were purified to homogeneity using a fluorescenceactivated cell sorter and were shown to retain hair inductive activityin a skin reconstitution assay. However, using flow cytometric analysisof GFP expression in DP cells immediately after isolation and after 90hours in culture, it was found that the inductive activity and GFPexpression of the DP cells were rapidly lost in culture. This suggeststhat a factor normally supplied by epidermal cells was required tomaintain DP cells in the anagen state.

Analysis of the Role of Shh in Anagen Phase

One candidate for the signal supplied by the epidermal cells was Shh,which is expressed in the epidermal component of the hair follicle andis required for the maturation of the dermal papilla during embryonicdevelopment. See, e.g., St. Jacques (1998) Curr. Biol. 8:1058-1068.During the hair cycle, exogenous Shh can accelerate the transition fromtelogen to anagen. Sato et al. (1999) J. Clin. Invest. 104:855-864.Expression of Shh, patched-1 (ptc-1), Gli-1 and the control of β-actingenes was analyzed in DP cells immediately after isolation and afterthree passages in culture. In skin from newborn transgenic miceundergoing active hair growth, Shh mRNA was detected in the GFP negativepopulation of sorted cells, which includes follicular epidermis, and wasabsent from the dermal papilla cells. Ptc-1 and Gli-1 are expressed inisolated DP but transcription levels decreased upon passage in culture.Transcriptional feedback in the Shh signal transduction cascade resultsin increased accumulation of mRNA encoding two of its components,patched (ptc) and Gli-1, in response to Shh signaling. This inductionserves as an indication of response to the Shh signal. Goodrich et al.(1996) Genes Dev. 10:301-312; Marigo et al. (1996) Development122:1225-1233. Message from both genes was readily detected in freshlysorted DP cells, but the abundance of both messages decreases toundetectable levels when isolated DP cells were cultured in the absenceof follicular epithelium. Thus, Shh signaling from follicular epitheliumto the dermal papilla occurs in the hair follicle and could cause DPactivation in anagen. Therefore, it was evaluated whether Shh signalingwas sufficient to rescue either GFP gene expression or hair inductiveactivity in DP cells maintained in culture. Freshly isolated DP cellswere co-cultured with CEFs expressing Shh or infected with a controlvector. The use of heterospecific feeder cells allowed analyzation ofgene expression in the DP cells by PCR with species-specific primers andconfirms that the DP cells received the Shh signal as demonstrated byinduction and maintenance of both ptc-1 and Gli-1, while expression ofboth genes is decreased in DP cells co-cultured with control feeders.β-actin sequences were used to normalize input cDNA and murine specificprimers were employed to discriminate between gene expression in the DPand feeder cell populations. However, as demonstrated by flow cytometricanalysis of DP cells co-cultures with feeder layers producing Wnt3 orShh or infected with control vectors, GFP expression declined atidentical rates in Shh treated and control populations. In addition, theratio of GFP positive and GFP negative cells was confirmed for eachtreatment. To confirm the correlation between GFP expression andmaintenance of the anagen state, these cells were mixed withkeratinocytes and grafted in bubble chambers on the backs of nude mousehosts. In this assay, the hairless skin which forms in the absence ofactive DP cells was observed whether or not the DP cells had beencultured in the presence of Shh. Both grafts of control or Shh treatedDP cells showed only occasional hairs three weeks after grafting to thenude mouse model. See Table 1.

As in vivo experiments have suggested that Shh stimulates the transitionfrom telogen to anagen, it was also assessed whether Shh signaling wassufficient to activate isolated DP cells which had been maintained inculture until they were formally analogous to telogen DP cells in thatboth GFP expression and hair inductive activity were lost. Again, nochange in either property was observed. Thus, although Shh may initiateanagen in vivo, it is likely that it acts on the DP in part indirectly,possibly by induction of a secondary signal in the epidermis.

Analysis of the Role of Wnt in Anagen Phase

A search of the sequence in the versican enhancer used to drive GFPexpression in the DP revealed a Lef/TCF binding motif and suggested thata Wnt might serve as the signal from the epidermis which activates theDP cells. This binding site appears to be required for GFP expressionbecause no GFP expression was observed in ten independent transgeniclines when this region was deleted from the expression construct.However, deletions from elsewhere in the construct had no effect on GFPexpression in transgenic mice. The Lef/TCF family of DNA bindingproteins mediates the transcriptional effects of Wnt signaling throughthe β-catenin pathway. Arias et al. (1999) Curr. Opin. Genet. & Dev.9:447-454. Wnts are secreted glycoproteins which bind to Frizzled. In aprocess dependent on disheveled proteins, Frizzled receptor engagementinhibits phosphorylation of β catenin by a complex including GSK3. Thisresults in accumulation of β-catenin protein in the cytoplasm andtranslocation to the nucleus where it can bind Lef/TCF and stimulatetranscription from associated genes. In freshly isolated DP cells,Frizzled-7 (frz-7), disheveled-2 (dsh-2), GSK3, β catenin, Lef1 and theGFP transgene are all expressed. Thus, the components of this signaltransduction cascade were expressed in freshly isolated DP cells. Afterthree passages, expression of frz-7, dsv-2 and Lef1 are reduced.Furthermore, Wnt3a is expressed in the follicular matrix cells andWnt3a, 5a and 7a transcripts were detected in the GFP negativepopulation from dissociated skin which includes the follicular epitheliafrom anagen hair follicles. Thus, all of these Wnts are candidates tomediate signaling to the dermal papilla.

Feeder cells expressing Wnts 3a, 4, 5a or 7a were used to test theeffects of Wnt signaling on freshly isolated DP cells. Co-culture withWnts3a or 7a resulted in maintenance of GFP fluorescence in the majorityof the DP cells as demonstrated by FACS analysis. RT PCR confirmed thatthis reflects increased levels of GFP RNA rather than stabilization ofthe encoded protein. In particular, GFP RNA levels are maintained byexposure to Wnt3a but not Shh or control feeder layers. Wnt 4 expressingcells also showed some maintenance of GFP expression but were lesspotent than either Wnt3a or 7a, while Wnt5a had no effect on DP GFPexpression. As suggested by GFP expression, the hair inductive activityof DP cells was also maintained in culture by Wnt signaling. When themurine DP cells were resorted, combined with keratinocytes and used toreconstitute skin on a nude mouse host, Wnt3a or 7a treated cells showeda dramatic increase in hair growth compared to cells co-cultured withcontrol feeders or Shh expression cells. The control and Shh treated DPcell grafts only showed an occasional hair three weeks after grafting,whereas DP cells exposed to Wnt3 a formed a dense patch of hair in thegraft. See Table 1.

While Wnt3a is sufficient to maintain DP cells in the anagen state, itcannot reactivate GFP expression or hair inductive activity in cellswhich have lost these properties in culture (data not shown). Analysisof transcripts encoding components of the Wnt signal transductioncascade reveals that Lef-1, disheveled-2 and Frizzled-7 are alldownregulated after maintenance in culture in the absence of Wnts andthis could explain the failure of exogenous Wnt to reactivate GFPexpression in these cells. Maintenance of expression of proteinsrequired for Wnt signal transduction by other factors expressed locallyin the epidermis could contribute to the coordination of morphogenesisin the follicle. The findings reported here suggest the possibility thata single Wnt expressed in the epidermis could coordinate development inboth the dermis and epidermis.

The results demonstrate that Wnt3a, and possibly other Wnts, expressedin the anagen hair follicle can act as inductive signals to maintain thedermal papilla in an anagen state. The results also suggest that Shhsignaling is not sufficient to initiate or maintain the anagen state ofDP cells, and may therefore act at least in part indirectly to promotethe transition to anagen in vivo. Finally these results extend thecorrelation between GFP expression in these transgenic DP cells andtheir ability to induce hair growth and thus suggest further use of thisapproach to dissect signaling between epithelia and mesenchyme tocoordinate morphogenesis.

Wnt Polypeptides and Nucleic Acid Sequences Encoding Wnt

Wnt polypeptides can be obtained in several ways including isolation ofWnt or expression of a sequence encoding Wnt by genetic engineeringmethods. The nucleotide sequences of various Wnt proteins from variousspecies are known. See, e.g., Gavin et al. (1990) Genes Dev.4:2319-2332; Lee et al. (1995) Proc. Natl Acad. Sci USA 92:2268-2272;and, Christiansen et al. (1995) Mech. Dev. 51:341-350 (describing, e.g.,murine Wnt1, Wnt2, Wnt3a, Wnt3b, Wnt4, Wnt5a, Wnt 5b, Wnt6, Wnt7a,Wnt7b, Wnt8a, Wnt8b, Wnt10b, Wnt11, Wnt12) and Vant Veer et al. (1984)Mol. Cell Biol. 4:2532-2534; Wainwright et al. (1988) EMBO J.7:1743-1748; and, PCT Publication WO 95/17416 (describing, e.g., humanWnt1, Wnt2, Wnt3, Wnt4, Wnt5a, Wnt7a and Wnt7b).

Analogs of Wnt or Other Proteins Involved in the Wnt-β-catenin SignalingPathway

Analogs can differ from naturally occurring protein in amino acidsequence or in ways that do not involve sequence, or both. Non-sequencemodifications include in vivo or in vitro chemical derivatization of theprotein. Non-sequence modifications include changes in acetylation,methylation, phosphorylation, carboxylation, or glycosylation.

Preferred analogs include a protein, e.g., Wnt, (or biologically activefragments thereof) whose sequences differ from the wild-type sequence byone or more conservative amino acid substitutions or by one or morenon-conservative amino acid substitutions, deletions, or insertionswhich do not abolish the biological activity. In a preferred embodiment,the sequence can differ from wild-type sequence by 1, 2, 3, 5, 10, butnot more than 20 to 30 amino acid residues. Conservative substitutionstypically include the substitution of one amino acid for another withsimilar characteristics, e.g., substitutions within the followinggroups: valine, glycine; glycine, alanine; valine, isoleucine, leucine;aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine. Other conservativesubstitutions can be taken from the table below.

TABLE 1 CONSERVATIVE AMINO ACID REPLACEMENTS For Amino Acid Code Replacewith any of Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine RD-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn,D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln AsparticAcid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys,S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu,D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln,D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, β-Ala Acp Isoleucine I D-Ile,Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Leu,D-Leu, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met,D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile, D-Ile,Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His,D-His, Trp, D-Trp, Trans-3,4, or 5-phenylproline, cis-3,4, or5-phenylproline Proline P D-Pro, L-I-thioazolidine-4- carboxylic acid,D-or L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr,allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr,Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val TyrosineY D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile,D-Ile, Met, D-Met

Other analogs within the invention are those with modifications whichincrease peptide stability; such analogs may contain, for example, oneor more non-peptide bonds (which replace the peptide bonds) in thepeptide sequence. Also included are: analogs that include residues otherthan naturally occurring L-amino acids, e.g., D-amino acids ornon-naturally occurring or synthetic amino acids, e.g., β or γ aminoacids; and cyclic analogs.

Production of Fragments and Analogs

Generation of Fragments

Fragments of a protein can be produced in several ways, e.g.,recombinantly, by proteolytic digestion, or by chemical synthesis.Internal or terminal fragments of a polypeptide can be generated byremoving one or more nucleotides from one end (for a terminal fragment)or both ends (for an internal fragment) of a nucleic acid which encodesthe polypeptide. Expression of the mutagenized DNA produces polypeptidefragments. Digestion with “end-nibbling” endonucleases can thus generateDNA's which encode an array of fragments. DNA's which encode fragmentsof a protein can also be generated by random shearing, restrictiondigestion or a combination of the above-discussed methods.

Fragments can also be chemically synthesized using techniques known inthe art such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, peptides of the present invention may bearbitrarily divided into fragments of desired length with no overlap ofthe fragments, or divided into overlapping fragments of a desiredlength.

Generation of Analogs: Production of Altered DNA and Peptide Sequencesby Random Methods

Amino acid sequence variants of a protein can be prepared by randommutagenesis of DNA which encodes a protein or a particular domain orregion of a protein. Useful methods include PCR mutagenesis andsaturation mutagenesis. A library of random amino acid sequence variantscan also be generated by the synthesis of a set of degenerateoligonucleotide sequences. (Methods for screening proteins in a libraryof variants are elsewhere herein.)

PCR Mutagenesis

In PCR mutagenesis, reduced Taq polymerase fidelity is used to introducerandom mutations into a cloned fragment of DNA (Leung et al., 1989,Technique 1:11-15). This is a very powerful and relatively rapid methodof introducing random mutations. The DNA region to be mutagenized isamplified using the polymerase chain reaction (PCR) under conditionsthat reduce the fidelity of DNA synthesis by Taq DNA polymerase, e.g.,by using a dGTP/dATP ratio of five and adding Mn²⁺ to the PCR reaction.The pool of amplified DNA fragments are inserted into appropriatecloning vectors to provide random mutant libraries.

Saturation Mutagenesis

Saturation mutagenesis allows for the rapid introduction of a largenumber of single base substitutions into cloned DNA fragments (Mayers etal., 1985, Science 229:242). This technique includes generation ofmutations, e.g., by chemical treatment or irradiation of single-strandedDNA in vitro, and synthesis of a complimentary DNA strand. The mutationfrequency can be modulated by modulating the severity of the treatment,and essentially all possible base substitutions can be obtained. Becausethis procedure does not involve a genetic selection for mutant fragmentsboth neutral substitutions, as well as those that alter function, areobtained. The distribution of point mutations is not biased towardconserved sequence elements.

Degenerate Oligonucleotides

A library of homologs can also be generated from a set of degenerateoligonucleotide sequences. Chemical synthesis of a degenerate sequencescan be carried out in an automatic DNA synthesizer, and the syntheticgenes then ligated into an appropriate expression vector. The synthesisof degenerate oligonucleotides is known in the art (see for example,Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) RecombinantDNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton,Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem.53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)Nucleic Acid Res. 11:477. Such techniques have been employed in thedirected evolution of other proteins (see, for example, Scott et al.(1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433;Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

Generation of Analogs: Production of Altered DNA and Peptide Sequencesby Directed Mutagenesis

Non-random or directed, mutagenesis techniques can be used to providespecific sequences or mutations in specific regions. These techniquescan be used to create variants which include, e.g., deletions,insertions, or substitutions, of residues of the known amino acidsequence of a protein. The sites for mutation can be modifiedindividually or in series, e.g., by (1) substituting first withconserved amino acids and then with more radical choices depending uponresults achieved, (2) deleting the target residue, or (3) insertingresidues of the same or a different class adjacent to the located site,or combinations of options 1-3.

Alanine Scanning Mutagenesis

Alanine scanning mutagenesis is a useful method for identification ofcertain residues or regions of the desired protein that are preferredlocations or domains for mutagenesis, Cunningham and Wells (Science244:1081-1085, 1989). In alanine scanning, a residue or group of targetresidues are identified (e.g., charged residues such as Arg, Asp, His,Lys, and Glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine). Replacement of an amino acidcan affect the interaction of the amino acids with the surroundingaqueous environment in or outside the cell. Those domains demonstratingfunctional sensitivity to the substitutions are then refined byintroducing further or other variants at or for the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to optimize the performance of amutation at a given site, alanine scanning or random mutagenesis may beconducted at the target codon or region and the expressed desiredprotein subunit variants are screened for the optimal combination ofdesired activity.

Oligonucleotide-mediated Mutagenesis

Oligonucleotide-mediated mutagenesis is a useful method for preparingsubstitution, deletion, and insertion variants of DNA, see, e.g.,Adelman et al., (DNA 2:183, 1983). Briefly, the desired DNA is alteredby hybridizing an oligonucleotide encoding a mutation to a DNA template,where the template is the single-stranded form of a plasmid orbacteriophage containing the unaltered or native DNA sequence of thedesired protein. After hybridization, a DNA polymerase is used tosynthesize an entire second complementary strand of the template thatwill thus incorporate the oligonucleotide primer, and will code for theselected alteration in the desired protein DNA. Generally,oligonucleotides of at least 25 nucleotides in length are used. Anoptimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al. (Proc.Natl. Acad. Sci. USA, 75: 5765[1978]).

Cassette Mutagenesis

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al. (Gene, 34:315[1985]). Thestarting material is a plasmid (or other vector) which includes theprotein subunit DNA to be mutated. The codon(s) in the protein subunitDNA to be mutated are identified. There must be a unique restrictionendonuclease site on each side of the identified mutation site(s). If nosuch restriction sites exist, they may be generated using theabove-described oligonucleotide-mediated mutagenesis method to introducethem at appropriate locations in the desired protein subunit DNA. Afterthe restriction sites have been introduced into the plasmid, the plasmidis cut at these sites to linearize it. A double-stranded oligonucleotideencoding the sequence of the DNA between the restriction sites butcontaining the desired mutation(s) is synthesized using standardprocedures. The two strands are synthesized separately and thenhybridized together using standard techniques. This double-strandedoligonucleotide is referred to as the cassette. This cassette isdesigned to have 3′ and 5′ ends that are comparable with the ends of thelinearized plasmid, such that it can be directly ligated to the plasmid.This plasmid now contains the mutated desired protein subunit DNAsequence.

Combinatorial Mutagenesis

Combinatorial mutagenesis can also be used to generate mutants. Forexample, the amino acid sequences for a group of homologs or otherrelated proteins are aligned, preferably to promote the highest homologypossible. All of the amino acids which appear at a given position of thealigned sequences can be selected to create a degenerate set ofcombinatorial sequences. The variegated library of variants is generatedby combinatorial mutagenesis at the nucleic acid level, and is encodedby a variegated gene library. For example, a mixture of syntheticoligonucleotides can be enzymatically ligated into gene sequences suchthat the degenerate set of potential sequences are expressible asindividual peptides, or alternatively, as a set of larger fusionproteins containing the set of degenerate sequences.

Primary High-through-put Methods for Screening Libraries of PeptideFragments or Homologs

Various techniques are known in the art for screening generated mutantgene products. Techniques for screening large gene libraries ofteninclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the genes under conditions in which detection of adesired activity, assembly into a trimeric molecules, binding to naturalligands, e.g., a Frz receptor or substrates, facilitates relatively easyisolation of the vector encoding the gene whose product was detected.Each of the techniques described below is amenable to high through-putanalysis for screening large numbers of sequences created, e.g., byrandom mutagenesis techniques.

Two Hybrid Systems

Two hybrid (interaction trap) assays can be used to identify a proteinthat interacts with Wnt. These may include agonists, superagonists, andantagonists. (The subject protein and a protein it interacts with areused as the bait protein and fish proteins.). These assays rely ondetecting the reconstitution of a functional transcriptional activatormediated by protein-protein interactions with a bait protein. Inparticular, these assays make use of chimeric genes which express hybridproteins. The first hybrid comprises a DNA-binding domain fused to thebait protein. e.g., a Wnt molecule or a fragment thereof. The secondhybrid protein contains a transcriptional activation domain fused to a“fish” protein, e.g. an expression library. If the fish and baitproteins are able to interact, they bring into close proximity theDNA-binding and transcriptional activator domains. This proximity issufficient to cause transcription of a reporter gene which is operablylinked to a transcriptional regulatory site which is recognized by theDNA binding domain, and expression of the marker gene can be detectedand used to score for the interaction of the bait protein with anotherprotein.

Display Libraries

In one approach to screening assays, the candidate peptides aredisplayed on the surface of a cell or viral particle, and the ability ofparticular cells or viral particles to bind an appropriate receptorprotein via the displayed product is detected in a “panning assay”. Forexample, the gene library can be cloned into the gene for a surfacemembrane protein of a bacterial cell, and the resulting fusion proteindetected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991)Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS 18:136-140).In a similar fashion, a detectably labeled ligand can be used to scorefor potentially functional peptide homologs. Fluorescently labeledligands, e.g., receptors, can be used to detect homolog which retainligand-binding activity. The use of fluorescently labeled ligands,allows cells to be visually inspected and separated under a fluorescencemicroscope, or, where the morphology of the cell permits, to beseparated by a fluorescence-activated cell sorter.

A gene library can be expressed as a fusion protein on the surface of aviral particle. For instance, in the filamentous phage system, foreignpeptide sequences can be expressed on the surface of infectious phage,thereby conferring two significant benefits. First, since these phagecan be applied to affinity matrices at concentrations well over 10¹³phage per milliliter, a large number of phage can be screened at onetime. Second, since each infectious phage displays a gene product on itssurface, if a particular phage is recovered from an affinity matrix inlow yield, the phage can be amplified by another round of infection. Thegroup of almost identical E. coli filamentous phages M13, fd., and f1are most often used in phage display libraries. Either of the phage gIIIor gVIII coat proteins can be used to generate fusion proteins withoutdisrupting the ultimate packaging of the viral particle. Foreignepitopes can be expressed at the NH₂-terminal end of pIII and phagebearing such epitopes recovered from a large excess of phage lackingthis epitope (Ladner et al. PCT publication WO 90/02909; Garrard et al.,PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem.267:16007-16010; Griffiths et al. (1993) EMBO J. 12:725-734; Clackson etal. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS89:4457-4461).

A common approach uses the maltose receptor of E. coli (the outermembrane protein, LamB) as a peptide fusion partner (Charbit et al.(1986) EMBO 5, 3029-3037). Oligonucleotides have been inserted intoplasmids encoding the LamB gene to produce peptides fused into one ofthe extracellular loops of the protein. These peptides are available forbinding to ligands, e.g., to antibodies, and can elicit an immuneresponse when the cells are administered to animals. Other cell surfaceproteins, e.g., OmpA (Schorr et al. (1991) Vaccines 91, pp. 387-392),PhoE (Agterberg, et al. (1990) Gene 88, 37-45), and PAL (Fuchs et al.(1991) Bio/Tech 9, 1369-1372), as well as large bacterial surfacestructures have served as vehicles for peptide display. Peptides can befused to pilin, a protein which polymerizes to form the pilus-a conduitfor interbacterial exchange of genetic information (Thiry et al. (1989)Appl. Environ. Microbiol. 55, 984-993). Because of its role ininteracting with other cells, the pilus provides a useful support forthe presentation of peptides to the extracellular environment. Anotherlarge surface structure used for peptide display is the bacterial motiveorgan, the flagellum. Fusion of peptides to the subunit proteinflagellin offers a dense array of may peptides copies on the host cells(Kuwajima et al. (1988) Bio/Tech. 6, 1080-1083). Surface proteins ofother bacterial species have also served as peptide fusion partners.Examples include the Staphylococcus protein A and the outer membraneprotease IgA of Neisseria (Hansson et al. (1992) J. Bacteriol. 174,4239-4245 and Klauser et al. (1990) EMBO J. 9, 1991-1999).

In the filamentous phage systems and the LamB system described above,the physical link between the peptide and its encoding DNA occurs by thecontainment of the DNA within a particle (cell or phage) that carriesthe peptide on its surface. Capturing the peptide captures the particleand the DNA within. An alternative scheme uses the DNA-binding proteinLacI to form a link between peptide and DNA (Cull et al. (1992) PNAS USA89:1865-1869). This system uses a plasmid containing the LacI gene withan oligonucleotide cloning site at its 3′-end. Under the controlledinduction by arabinose, a LacI-peptide fusion protein is produced. Thisfusion retains the natural ability of LacI to bind to a short DNAsequence known as LacO operator (LacO). By installing two copies of LacOon the expression plasmid, the LacI-peptide fusion binds tightly to theplasmid that encoded it. Because the plasmids in each cell contain onlya single oligonucleotide sequence and each cell expresses only a singlepeptide sequence, the peptides become specifically and stably associatedwith the DNA sequence that directed its synthesis. The cells of thelibrary are gently lysed and the peptide-DNA complexes are exposed to amatrix of immobilized receptor to recover the complexes containingactive peptides. The associated plasmid DNA is then reintroduced intocells for amplification and DNA sequencing to determine the identity ofthe peptide ligands. As a demonstration of the practical utility of themethod, a large random library of dodecapeptides was made and selectedon a monoclonal antibody raised against the opioid peptide dynorphin B.A cohort of peptides was recovered, all related by a consensus sequencecorresponding to a six-residue portion of dynorphin B. (Cull et al.(1992) Proc. Natl. Acad. Sci U.S.A. 89-1869)

This scheme, sometimes referred to as peptides-on-plasmids, differs intwo important ways from the phage display methods. First, the peptidesare attached to the C-terminus of the fusion protein, resulting in thedisplay of the library members as peptides having free carboxy termini.Both of the filamentous phage coat proteins, pIII and pVIII, areanchored to the phage through their C-termini, and the guest peptidesare placed into the outward-extending N-terminal domains. In somedesigns, the phage-displayed peptides are presented right at the aminoterminus of the fusion protein. (Cwirla, et al. (1990) Proc. Natl. Acad.Sci. U.S.A. 87, 6378-6382) A second difference is the set of biologicalbiases affecting the population of peptides actually present in thelibraries. The LacI fusion molecules are confined to the cytoplasm ofthe host cells. The phage coat fusions are exposed briefly to thecytoplasm during translation but are rapidly secreted through the innermembrane into the periplasmic compartment, remaining anchored in themembrane by their C-terminal hydrophobic domains, with the N-termini,containing the peptides, protruding into the periplasm while awaitingassembly into phage particles. The peptides in the LacI and phagelibraries may differ significantly as a result of their exposure todifferent proteolytic activities. The phage coat proteins requiretransport across the inner membrane and signal peptidase processing as aprelude to incorporation into phage. Certain peptides exert adeleterious effect on these processes and are underrepresented in thelibraries (Gallop et al. (1994) J. Med. Chem. 37(9):1233-1251). Theseparticular biases are not a factor in the LacI display system.

The number of small peptides available in recombinant random librariesis enormous. Libraries of 10⁷-10⁹ independent clones are routinelyprepared. Libraries as large as 10¹¹ recombinants have been created, butthis size approaches the practical limit for clone libraries. Thislimitation in library size occurs at the step of transforming the DNAcontaining randomized segments into the host bacterial cells. Tocircumvent this limitation, an in vitro system based on the display ofnascent peptides in polysome complexes has recently been developed. Thisdisplay library method has the potential of producing libraries 3-6orders of magnitude larger than the currently available phage/phagemidor plasmid libraries. Furthermore, the construction of the libraries,expression of the peptides, and screening, is done in an entirelycell-free format.

In one application of this method (Gallop et al. (1994) J. Med. Chem.37(9):1233-1251), a molecular DNA library encoding 10¹² decapeptides wasconstructed and the library expressed in an E. coli S30 in vitro coupledtranscription/translation system. Conditions were chosen to stall theribosomes on the mRNA, causing the accumulation of a substantialproportion of the RNA in polysomes and yielding complexes containingnascent peptides still linked to their encoding RNA. The polysomes aresufficiently robust to be affinity purified on immobilized receptors inmuch the same way as the more conventional recombinant peptide displaylibraries are screened. RNA from the bound complexes is recovered,converted to cDNA, and amplified by PCR to produce a template for thenext round of synthesis and screening. The polysome display method canbe coupled to the phage display system. Following several rounds ofscreening, cDNA from the enriched pool of polysomes was cloned into aphagemid vector. This vector serves as both a peptide expression vector,displaying peptides fused to the coat proteins, and as a DNA sequencingvector for peptide identification. By expressing the polysome-derivedpeptides on phage, one can either continue the affinity selectionprocedure in this format or assay the peptides on individual clones forbinding activity in a phage ELISA, or for binding specificity in acompletion phage ELISA (Barret, et al. (1992) Anal. Biochem204,357-364). To identify the sequences of the active peptides onesequences the DNA produced by the phagemid host.

Secondary Screens

The high through-put assays described above can be followed by secondaryscreens in order to identify further biological activities which will,e.g., allow one skilled in the art to differentiate agonists fromantagonists. The type of a secondary screen used will depend on thedesired activity that needs to be tested. For example, an assay can bedeveloped in which the ability to inhibit an interaction between aprotein of interest and its respective ligand can be used to identifyantagonists from a group of peptide fragments isolated though one of theprimary screens described above.

Therefore, methods for generating fragments and analogs and testing themfor activity are known in the art. Once the core sequence of interest isidentified, it is routine to perform for one skilled in the art toobtain analogs and fragments.

Peptide Mimetics

The invention also provides for reduction of the protein binding domainsof the subject Wnt polypeptides to generate mimetics, e.g. peptide ornon-peptide agents. See, for example, “Peptide inhibitors of humanpapillomavirus protein binding to retinoblastoma gene protein” Europeanpatent applications EP-412,762A and EP-B31,080A.

Non-hydrolyzable peptide analogs of critical residues can be generatedusing benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistryand Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry andBiology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988), substituted gama lactam rings (Garvey et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al. (1986)J. Med Chem 29:295; and Ewenson et al. in Peptides: Structure andFunction (Proceedings of the 9th American Peptide Symposium) PierceChemical Co. Rockland, Ill., 1985), β-turn dipeptide cores (Nagai et al.(1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J. Chem SocPerkin Trans 1:1231), and β-aminoalcohols (Gordon et al. (1985) BiochemBiophys Res Commun 126:419; and Dann et al. (1986) Biochem Biophys ResCommun 134:71).

Fusion Proteins

Polypeptides for modulating the level of Wnt protein can be fused toanother protein or portion thereof. For example, a Wnt protein orportion thereof, such as the Frizzled binding portion of Wnt, can beoperably linked to another polypeptide moiety to enhance solubility.Examples of a protein which can be fused with Wnt or portions thereofinclude a plasma protein or fragment thereof, which can improve thecirculating half life of Wnt. For example, the fusion protein can be aWnt-immunoglobulin (Ig) fusion protein in which the Wnt sequence isfused to a sequence derived from the immunoglobulin superfamily. Severalsoluble fusion protein constructs have been disclosed wherein theextracellular domain of a cell surface glycoprotein is fused with theconstant F(c) region of an immunoglobulin. For example, Capon et al.(1989) Nature 337(9):525-531, provide guidance on generating a longerlasting CD4 analog by fusing CD4 to an immunoglobulin (IgG1). See also,Capon et al., U.S. Pat. Nos. 5,116,964 and 5,428,130 (CD4-IgG fusionconstructs); Linsley et al., U.S. Pat. No. 5,434,131 (CTLA4-IgG1 andB7-IgG1 fusion constructs); Linsley et al. (1991) J. Exp. Med174:561-569 (CTLA4-IgG1 fusion constructs); and Linsley et al. (1991) J.Exp. Med 173:721-730 (CD28-IgG1 and B7-IgG1 fusion constructs). Suchfusion proteins have proven useful for modulating receptor-ligandinteractions and reducing inflammation in vivo. For example, fusionproteins in which an extracellular domain of cell surface tumor necrosisfactor receptor (TNFR) proteins has been fused to an immunoglobulinconstant (Fc) region have been used in vivo. See, for example, Morelandet al. (1997) N. Engl. J. Med. 337(3):141-147; and, van der Poll et al.(1997) Blood 89(10):3727-3734).

Antibodies

The invention also includes antibodies specifically reactive with asubject Wnt polypeptides or Frizzled as well as antibodies specificallyreactive with other proteins of the Wnt-β-catenin signaling pathway,e.g., intrabodies. Anti-protein/anti-peptide antisera or monoclonalantibodies can be made as described herein by using standard protocols(See, for example, Antibodies: A Laboratory Manual ed. by Harlow andLane (Cold Spring Harbor Press: 1988)).

A Wnt protein, or a portion or fragment thereof, can be used as animmunogen to generate antibodies that bind Wnt using standard techniquesfor polyclonal and monoclonal antibody preparation. The full-length Wntprotein can be used or, alternatively, antigenic peptide fragments ofWnt can be used as immunogens.

Typically, Wnt or a Wnt peptide is used to prepare antibodies byimmunizing a suitable subject, (e.g., rabbit, goat, mouse or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for example, a recombinant Wnt peptide, or a chemicallysynthesized Wnt peptide. See, e.g., U.S. Pat. No. 5,460,959; andco-pending U.S. application Ser. No. 08/334,797; U.S. Ser. No.08/231,439; U.S. Ser. No. 08/334,455; and U.S. Ser. No. 08/928,881 whichare hereby expressly incorporated by reference in their entirety. Thenucleotide and amino acid sequences of Wnt are known and described, forexample, in Vant Veer et al. (1984) Mol. Cell. Biol. 4:2532-2534, Gavinet al. (1992) Gene Dev. 4:2319-2332; Lee et al. (1995) Proc. Natl Acad.Sci USA 92:2268-2272; Christiansen et al. (1995) Mech. Dev. 51:341-350,Wainwright et al. (1988) EMBO J. 7:1743-1748; and, PCT Publication WO95/17416. The nucleotide and amino acid sequence of other members of theWnt-β-catenin signal pathway are also known. The preparation can furtherinclude an adjuvant, such as Freund's complete or incomplete adjuvant,or similar immunostimulatory agent. Immunization of a suitable subjectwith an immunogenic Wnt preparation induces a polyclonal anti-Wntantibody response.

Anti-Wnt antibodies or fragments thereof can be used to inhibit thelevels of Wnt protein. Examples of anti-Wnt antibody fragments includeF(v), Fab, Fab′ and F(ab′)₂ fragments which can be generated by treatingthe antibody with an enzyme such as pepsin. The term “monoclonalantibody” or “monoclonal antibody composition”, as used herein, refersto a population of antibody molecules that contain only one species ofan antigen binding site capable of immunoreacting with a particularepitope of Wnt. A monoclonal antibody composition thus typicallydisplays a single binding affinity for a particular Wnt protein withwhich it immunoreacts.

Additionally, anti-Wnt antibodies produced by genetic engineeringmethods, such as chimeric and humanized monoclonal antibodies,comprising both human and non-human portions, which can be made usingstandard recombinant DNA techniques, can be used. Such chimeric andhumanized monoclonal antibodies can be produced by genetic engineeringusing standard DNA techniques known in the art, for example usingmethods described in Robinson et al. International Application No.PCT/US86/02269; Akira, et al. European Patent Application 184,187;Taniguchi, M., European Patent Application 171,496; Morrison et al.European Patent Application 173,494; Neuberger et al. PCT InternationalPublication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567;Cabilly et al. European Patent Application 125,023; Better et al.,Science 240:1041-1043, 1988; Liu et al., PNAS 84:3439-3443, 1987; Liu etal., J. Immunol. 139:3521-3526, 1987; Sun et al. PNAS 84:214-218, 1987;Nishimura et al., Canc. Res. 47:999-1005, 1987; Wood et al., Nature314:446-449, 1985; and Shaw et al., J. Natl. Cancer Inst. 80:1553-1559,1988); Morrison, S. L., Science 229:1202-1207, 1985; Oi et al.,BioTechniques 4:214, 1986; Winter U.S. Pat. No. 5,225,539; Jones et al.,Nature 321:552-525, 1986; Verhoeyan et al., Science 239:1534, 1988; andBeidler et al., J. Immunol. 141:4053-4060, 1988.

In addition, a human monoclonal antibody directed against Wnt can bemade using standard techniques. For example, human monoclonal antibodiescan be generated in transgenic mice or in immune deficient miceengrafted with antibody-producing human cells. Methods of generatingsuch mice are describe, for example, in Wood et al. PCT publication WO91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg etal. PCT publication WO 92/03918; Kay et al. PCT publication WO 92/03917;Kay et al. PCT publication WO 93/12227; Kay et al. PCT publication94/25585; Rajewsky et al. Pct publication WO 94/04667; Ditullio et al.PCT publication WO 95/17085; Lonberg, N. et al. (1994) Nature368:856-859; Green, L. L. et al. (1994) Nature Genet. 7:13-21; Morrison,S. L. et al. (1994) Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggemanet al. (1993) Year Immunol 7:33-40; Choi et al. (1993) Nature Genet.4:117-123; Tuaillon et al. (1993) PNAS 90:3720-3724; Bruggeman et al.(1991) Eur J. Immunol 21:1323-1326); Duchosal et al. PCT publication WO93/05796; U.S. Pat. No. 5,411,749; McCune et al. (1988) Science241:1632-1639), Kamel-Reid et al. (1988) Science 242:1706; Spanopoulou(1994) Genes & Development 8:1030-1042; Shinkai et al. (1992) Cell68:855-868). A human antibody-transgenic mouse or an immune deficientmouse engrafted with human antibody-producing cells or tissue can beimmunized with Wnt or an antigenic Wnt peptide and splenocytes fromthese immunized mice can then be used to create hybridomas. Methods ofhybridoma production are well known.

Human monoclonal antibodies against Wnt can also be prepared byconstructing a combinatorial immunoglobulin library, such as a Fab phagedisplay library or a scFv phage display library, using immunoglobulinlight chain and heavy chain cDNAs prepared from mRNA derived fromlymphocytes of a subject. See, e.g., McCafferty et al. PCT publicationWO 92/01047; Marks et al. (1991) J. Mol. Biol. 222:581-597; and Griffthset al. (1993) EMBO J. 12:725-734. In addition, a combinatorial libraryof antibody variable regions can be generated by mutating a known humanantibody. For example, a variable region of a human antibody known tobind Wnt, can be mutated, by for example using randomly alteredmutagenized oligonucleotides, to generate a library of mutated variableregions which can then be screened to bind to Wnt. Methods of inducingrandom mutagenesis within the CDR regions of immunoglobin heavy and/orlight chains, methods of crossing randomized heavy and light chains toform pairings and screening methods can be found in, for example, Barbaset al. PCT publication WO 96/07754; Barbas et al. (1992) Proc. Nat'lAcad. Sci. USA 89:4457-4461.

The immunoglobulin library can be expressed by a population of displaypackages, preferably derived from filamentous phage, to form an antibodydisplay library. Examples of methods and reagents particularly amenablefor use in generating antibody display library can be found in, forexample, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTpublication WO 92/18619; Dower et al. PCT publication WO 91/17271;Winter et al. PCT publication WO 92/20791; Markland et al. PCTpublication WO 92/15679; Breitling et al. PCT publication WO 93/01288;McCafferty et al. PCT publication WO 92/01047; Garrard et al. PCTpublication WO 92/09690; Ladner et al. PCT publication WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) HumAntibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffths et al. (1993) supra; Hawkins et al. (1992) J. Mol Biol226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) PNAS 88:7978-7982. Once displayed on the surface ofa display package (e.g., filamentous phage), the antibody library isscreened to identify and isolate packages that express an antibody thatbinds Wnt. In a preferred embodiment, the primary screening of thelibrary involves panning with an immobilized Wnt and display packagesexpressing antibodies that bind immobilized Wnt are selected.

Antisense Wnt Nucleic Acid Sequences

Nucleic acid molecules which are antisense to a nucleotide encoding Wntcan be used as an agent which inhibits Wnt expression. An “antisense”nucleic acid includes a nucleotide sequence which is complementary to a“sense” nucleic acid encoding Wnt, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. Accordingly, an antisense nucleic acid can form hydrogen bondswith a sense nucleic acid. The antisense nucleic acid can becomplementary to an entire Wnt coding strand, or to only a portionthereof. For example, an antisense nucleic acid molecule which antisenseto the “coding region” of the coding strand of a nucleotide sequenceencoding Wnt can be used.

The coding strand sequences encoding Wnt are known. For example, atleast 7 Wnt genes have been identified in human (Wnt-1, 2, 3, 4, 5a, 7aand 7b). See, e.g., Vant Veer et al. (1984) Mol. Cell. Biol.4:2532-2534, Gavin et al. (1992) Gene Dev. 4:2319-2332; Lee et al.(1995) Proc. Natl Acad. Sci USA 92:2268-2272; Christiansen et al. (1995)Mech. Dev. 51:341-350, Wainwright et al. (1988) EMBO J. 7:1743-1748;and, PCT Publication WO 95/17416. Given the coding strand sequencesencoding Wnt, antisense nucleic acids can be designed according to therules of Watson and Crick base pairing. The antisense nucleic acidmolecule can be complementary to the entire coding region of Wnt mRNA,but more preferably is an oligonucleotide which is antisense to only aportion of the coding or noncoding region of Wnt mRNA. For example, theantisense oligonucleotide can be complementary to the region surroundingthe translation start site of Wnt mRNA. An antisense oligonucleotide canbe, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50nucleotides in length. An antisense nucleic acid can be constructedusing chemical synthesis and enzymatic ligation reactions usingprocedures known in the art. For example, an antisense nucleic acid(e.g., an antisense oligonucleotide) can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest.

Gene Therapy

The gene constructs of the invention can also be used as a part of agene therapy protocol to deliver nucleic acids encoding either anagonistic or antagonistic form of a Wnt polypeptide. The inventionfeatures expression vectors for in vivo transfection and expression of aWnt polypeptide in particular cell types so as to reconstitute thefunction of, or alternatively, antagonize the function of a Wntpolypeptide in a cell in which that polypeptide is misexpressed.Expression constructs of Wnt polypeptides, may be administered in anybiologically effective carrier, e.g. any formulation or compositioncapable of effectively delivering the Wnt gene to cells in vivo.Approaches include insertion of the subject gene in viral vectorsincluding recombinant retroviruses, adenovirus, adeno-associated virus,and herpes simplex virus-1, or recombinant bacterial or eukaryoticplasmids. Viral vectors transfect cells directly; plasmid DNA can bedelivered with the help of, for example, cationic liposomes (lipofectin)or derivatized (e.g. antibody conjugated), polylysine conjugates,gramacidin S, artificial viral envelopes or other such intracellularcarriers, as well as direct injection of the gene construct or CaPO₄precipitation carried out in vivo.

A preferred approach for in vivo introduction of nucleic acid into acell is by use of a viral vector containing nucleic acid, e.g. a cDNA,encoding a Wnt polypeptide. Infection of cells with a viral vector hasthe advantage that a large proportion of the targeted cells can receivethe nucleic acid. Additionally, molecules encoded within the viralvector, e.g., by a cDNA contained in the viral vector, are expressedefficiently in cells which have taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors can be used as arecombinant gene delivery system for the transfer of exogenous genes invivo, particularly into humans. These vectors provide efficient deliveryof genes into cells, and the transferred nucleic acids are stablyintegrated into the chromosomal DNA of the host. The development ofspecialized cell lines (termed “packaging cells”) which produce onlyreplication-defective retroviruses has increased the utility ofretroviruses for gene therapy, and defective retroviruses arecharacterized for use in gene transfer for gene therapy purposes (for areview see Miller, A. D. (1990) Blood 76:271). A replication defectiveretrovirus can be packaged into virions which can be used to infect atarget cell through the use of a helper virus by standard techniques.Protocols for producing recombinant retroviruses and for infecting cellsin vitro or in vivo with such viruses can be found in Current Protocolsin Molecular Biology, Ausubel, F. M. et al. (eds.) Greene PublishingAssociates, (1989), Sections 9.10-9.14 and other standard laboratorymanuals. Examples of suitable retroviruses include pLJ, pZIP, pWE andpEM which are known to those skilled in the art. Examples of suitablepackaging virus lines for preparing both ecotropic and amphotropicretroviral systems include ψCrip, ψCre, ψ2 and ψAm. Retroviruses havebeen used to introduce a variety of genes into many different celltypes, including epithelial cells, in vitro and/or in vivo (see forexample Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan(1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988)Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc.Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad.Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

Another viral gene delivery system useful in the present inventionutilizes adenovirus-derived vectors. The genome of an adenovirus can bemanipulated such that it encodes and expresses a gene product ofinterest but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See, for example, Berkner et al. (1988)BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; andRosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 dl324 or other strains ofadenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known to those skilled in theart. Recombinant adenoviruses can be advantageous in certaincircumstances in that they are not capable of infecting nondividingcells and can be used to infect a wide variety of cell types, includingepithelial cells (Rosenfeld et al. (1992) cited supra). Furthermore, thevirus particle is relatively stable and amenable to purification andconcentration, and as above, can be modified so as to affect thespectrum of infectivity. Additionally, introduced adenoviral DNA (andforeign DNA contained therein) is not integrated into the genome of ahost cell but remains episomal, thereby avoiding potential problems thatcan occur as a result of insertional mutagenesis in situ whereintroduced DNA becomes integrated into the host genome (e.g., retroviralDNA). Moreover, the carrying capacity of the adenoviral genome forforeign DNA is large (up to 8 kilobases) relative to other gene deliveryvectors (Berkner et al. cited supra; Haj-Ahmand and Graham (1986) J.Virol. 57:267).

Yet another viral vector system useful for delivery of the subject geneis the adeno-associated virus (AAV). Adeno-associated virus is anaturally occurring defective virus that requires another virus, such asan adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka etal. (1992) Curr. Topics in Micro. and Immunol. 158:97-129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356;Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al.(1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of a Wntpolypeptide in the tissue of an animal. Most nonviral methods of genetransfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In preferredembodiments, non-viral gene delivery systems of the present inventionrely on endocytic pathways for the uptake of the subject Wnt gene by thetargeted cell. Exemplary gene delivery systems of this type includeliposomal derived systems, poly-lysine conjugates, and artificial viralenvelopes.

In a representative embodiment, a gene encoding a Wnt polypeptide can beentrapped in liposomes bearing positive charges on their surface (e.g.,lipofectins) and (optionally) which are tagged with antibodies againstcell surface antigens of the target tissue (Mizuno et al. (1992) NoShinkei Geka 20:547-551; PCT publication WO91/06309; Japanese patentapplication 1047381; and European patent publication EP-A-43075).

In clinical settings, the gene delivery systems for the therapeutic Wntgene can be introduced into a patient by any of a number of methods,each of which is familiar in the art. For instance, a pharmaceuticalpreparation of the gene delivery system can be introduced systemically,e.g. by intravenous injection, and specific transduction of the proteinin the target cells occurs predominantly from specificity oftransfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al.(1994) PNAS 91: 3054-3057).

The pharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery system can beproduced in tact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system.

In other preferred embodiments, ex vivo gene therapy approaches can beused.

Cell Therapy

Wnt can also be increased in a subject by introducing into a cell, e.g.,a fibroblast, keratinocyte, epithelial cell, e.g., a hair follicle cell,e.g., a DP cell, a nucleotide sequence that modulates the production ofWnt, e.g., a nucleotide sequence encoding a Wnt polypeptide orfunctional fragment or analog thereof, a promoter sequence, e.g., apromoter sequence from a Wnt gene or from another gene; an enhancersequence, e.g., 5′ untranslated region (UTR), e.g., a 5′ UTR from a Wntgene or from another gene, a 3′ UTR, e.g., a 3′ UTR from a Wnt gene orfrom another gene; a polyadenylation site; an insulator sequence; oranother sequence that modulates the expression of Wnt. The cell can thenbe introduced into the subject.

Primary and secondary cells to be genetically engineered can be obtainedfrom a variety of tissues and include cell types which can be maintainedpropagated in culture. For example, primary and secondary cells includefibroblasts, keratinocytes, epithelial cells (e.g., DP cells),endothelial cells, glial cells, neural cells, formed elements of theblood (e.g., lymphocytes, bone marrow cells), muscle cells (myoblasts)and precursors of these somatic cell types. Primary cells are preferablyobtained from the individual to whom the genetically engineered primaryor secondary cells are administered. However, primary cells may beobtained for a donor (other than the recipient) of the same species oranother species (e.g., mouse, rat, rabbit, cat, dog, pig, cow, bird,sheep, goat, horse).

The term “primary cell” includes cells present in a suspension of cellsisolated from a vertebrate tissue source (prior to their being platedi.e., attached to a tissue culture substrate such as a dish or flask),cells present in an explant derived from tissue, both of the previoustypes of cells plated for the first time, and cell suspensions derivedfrom these plated cells. The term “secondary cell” or “cell strain”refers to cells at all subsequent steps in culturing. That is, the firsttime a plated primary cell is removed from the culture substrate andreplated (passaged), it is referred to herein as a secondary cell, asare all cells in subsequent passages. Secondary cells are cell strainswhich consist of secondary cells which have been passaged one or moretimes. A cell strain consists of secondary cells that: 1) have beenpassaged one or more times; 2) exhibit a finite number of meanpopulation doublings in culture; 3) exhibit the properties ofcontact-inhibited, anchorage dependent growth (anchorage-dependence doesnot apply to cells that are propagated in suspension culture); and 4)are not immortalized. A “clonal cell strain” is defined as a cell strainthat is derived from a single founder cell. A “heterogenous cell strain”is defined as a cell strain that is derived from two or more foundercells.

Primary or secondary cells of vertebrate, particularly mammalian, origincan be transfected with an exogenous nucleic acid sequence whichincludes a nucleic acid sequence encoding a signal peptide, and/or aheterologous nucleic acid sequence, e.g., encoding Wnt, and produce theencoded product stably and reproducibly in vitro and in vivo, overextended periods of time. A heterologous amino acid can also be aregulatory sequence, e.g., a promoter, which causes expression, e.g.,inducible expression or upregulation, of an endogenous Wnt sequence. Anexogenous nucleic acid sequence can be introduced into a primary orsecondary cell by homologous recombination as described, for example, inU.S. Pat. No. 5,641,670, the contents of which are incorporated hereinby reference.

The transfected primary or secondary cells may also include DNA encodinga selectable marker which confers a selectable phenotype upon them,facilitating their identification and isolation. Methods for producingtransfected primary and secondary cells which stably express exogenoussynthetic DNA, clonal cell strains and heterogeneous cell strains ofsuch transfected cells, methods of producing the clonal heterogeneouscell strains, and methods of treating or preventing an abnormal orundesirable condition through the use of populations of transfectedprimary or secondary cells are part of the present invention.

Transfection of Primary or Secondary Cells of Clonal or HeterogeneousCell Strains

Vertebrate tissue can be obtained by standard methods such a punchbiopsy or other surgical methods of obtaining a tissue source of theprimary cell type of interest. For example, punch biopsy or removal of ahair follicle is used to obtain a source of fibroblasts, keratinocytes,or endothelial cells, e.g., hair follicle cells or DP cells. A mixtureof primary cells is obtained from the tissue, using known methods, suchas enzymatic digestion or explanting. If enzymatic digestion is used,enzymes such as collagenase, hyaluronidase, dispase, pronase, trypsin,elastase and chymotrypsin can be used.

The resulting primary cell mixture can be transfected directly or it canbe cultured first, removed from the culture plate and resuspended beforetransfection is carried out. Primary cells or secondary cells arecombined with exogenous nucleic acid sequence to, e.g., stably integrateinto their genomes, and treated in order to accomplish transfection. Theexogenous nucleic acid sequence can optionally include DNA encoding aselectable marker. The exogenous nucleic acid sequence and selectablemarker-encoding DNA can either be on separate constructs or on a singleconstruct. An appropriate quantity of DNA is used to ensure that atleast one stably transfected cell containing and appropriatelyexpressing exogenous DNA is produced. In general, approximately 0.1 to500 μg of DNA is used.

As used herein, the term “transfection” includes a variety of techniquesfor introducing an exogenous nucleic acid into a cell including calciumphosphate or calcium chloride precipitation, microinjection,DEAE-dextrin-mediated transfection, lipofection or electrophoration.

Electroporation is carried out at approximate voltage and capacitance(and corresponding time constant) to result in entry of the DNAconstruct(s) into the primary or secondary cells. Electroporation can becarried out over a wide range of voltages (e.g., 50 to 2000 volts) andcorresponding capacitance. Total DNA of approximately 0.1 to 500 μg isgenerally used.

Methods such as calcium phosphate precipitation, modified calciumphosphate precipitation an polybrene precipitation, liposome fusion andreceptor-mediated gene delivery can also be used to transect cells.Primary or secondary cells can also be transfected using microinjection.A stably, transfected cell can then be isolated and cultured and subcultivated, under culturing conditions and for sufficient time topropagate stably transfected secondary cells an produce a clonal cellstrain of transfected secondary cells. Alternatively, more than onetransfected cell is cultured and sub cultured, resulting in productionof a heterogeneous cell strain.

Transfected primary or secondary cells undergo sufficient numberdoubling to produce either a clonal cell strain or a heterogeneous cellstrain of sufficient size to provide the therapeutic protein to anindividual in effective amounts. In general, for example, 0.1 cm² ofskin is biopsies and assumed to contain 1,000,000 cells; one cell isused to produce a clonal cell strain and undergoes approximately 27doublings to produce 100 million transfected secondary cells. If aheterogeneous cell strain is to be produced from an original transfectedpopulation of approximately 1000,000 cells, only 10 doublings are neededto produce 100 million transfected cells.

The number of required cells in a transfected clonal heterogeneous cellstrain is variable and depends on a variety of factors, including butnot limited to, the use of the transfected cells, the functional levelof the exogenous DNA in the transfected cells, the site of implantationof the transfected cells (for example, the number of cells that can beused is limited by the anatomical site of implantation), and the age,surface area, and clinical condition of the patient. The put thesefactors in perspective, to deliver therapeutic levels of human growthhormone in an otherwise healthy 10 kg patient with isolated growthhormone deficiency, approximately one to five hundred milliontransfected fibroblast would be necessary (the volume of these cells isabout that of the very tip of the patient's thumb).

Implantation of Clonal Cell Strain or Heterogeneous Cell Strain ofTransfected Secondary Cells

The transfected cells, e.g., cells produced as described herein, can beintroduced into an individual to whom the product is to be delivered.The clonal cell strain or heterogeneous cell strain is then introducedinto an individual. Various routed of administration and various sites(e.g., renal sub capsular, subcutaneous, central nervous system(including intrathecal), intravascular, intrahepatic, intrasplanchnic,intraperitoneal (including intraomental), intramuscularly implantation)can be used. One implanted in individual, the transfected cells producethe product encoded by the heterologous DNA or are affected by theheterologous DNA itself. For example, an individual who suffers from acondition related to unwanted angiogenesis is a candidate forimplantation of Wnt producing cells.

The individual can have a small skin biopsy performed; this is a simpleprocedure which can be performed on an outpatient basis. The piece ofskin is taken, for example, from under the arm and can require about oneminute to remove. The sample is processed, resulting in isolation of thepatient's cell (e.g., fibroblasts) and genetically engineered to produceWnt or another protein or molecule that induces the production of Wnt.Based on the age, weight, and clinical condition of the patient, therequired number of cells are grown in large-scale culture. The entireprocess should require 4-6 weeks and, at the end of that time, theappropriate number of genetically engineered cells are introduced intothe individual, once again as an outpatient (e.g., by injecting themback under the patient's skin, e.g., on the scalp or face). The patientis now capable of producing Wnt which can ameliorate symptoms of hairloss.

For some, this will be a one-time treatment and, for others, multiplecell therapy treatments will be required.

As this example suggests, the cells used will generally bepatient-specific genetically engineered cells. It is possible, however,to obtain cells from another individual of the same species or from adifferent species. Use of such cells might require administration of animmunosuppressant, alteration of histocompatibility antigens, or use ofa barrier device to prevent rejection of the implanted cells.

Transfected primary or secondary cells can be administered alone or inconjunction with a barrier or agent for inhibiting immune responseagainst the cell in a recipient subject. For example, animmunosuppressive agent can be administered to a subject to inhibit orinterfere with normal response in the subject. Preferably, theimmunosuppressive agent is an immunosuppressive drug which inhibits Tcell/or B cell activity in a subject. Examples of such immunosuppressivedrugs commercially available (e.g., cyclosporin A is commerciallyavailable from Sandoz Corp. East Hanover, N.J.).

An immunosuppressive agent, e.g., drug, can be administered to a subjectat a dosage sufficient to achieve the desired therapeutic effect (e.g.,inhibition of rejection of the cells). Dosage ranges forimmunosuppressive drugs are known in the art. See, e.g., Freed et al.(1992) N. Engl. J. Med. 327:1549; Spencer et al. (1992) N. Engl. J. Med.327:1541' Widner et al. (1992) n. Engl. J. Med. 327:1556). Dosage valuesmay vary according to factors such as the disease state, age, sex, andweight of the individual.

Another agent with can be used to inhibit T cell activity in a subjectis an antibody, or fragment of derivative thereof. Antibodies capable ofdepleting or sequestering T cells in vivo are known in the art.Polyclonal antisera can be used, for example, anti-lymphocyte serum.Alternatively, one or more monoclonal antibodies can be used. PreferredT cell depleting antibodies include monoclonal antibodies which bind toCD2, CD3, CD4, CD8, CD40, CD40, ligand on the cell surface. Suchantibodies are known in the art and are commercially available, forexample, from American Type Culture Collection. A preferred antibody forbinding CD3 on human T cells is OKT3 (ATCC CRL 8001).

An antibody which depletes, sequesters or inhibits T cells within arecipient subject can be administered in a dose for an appropriate timeto inhibit rejection of cells upon transplantation. Antibodies arepreferably administered intravenously in a pharmaceutically acceptablecarrier of diluent (e.g., saline solution).

An advantage of the use of transfected or secondary cells is that bycontrolling the number of cells introduced into an individual, one cancontrol the amount of the protein delivered to the body. In addition, insome cases, it is possible to remove the transfected cells of there isno longer a need for the product. A further advantage of treatment byuse of transfected primary or secondary cells of the present inventionis that production of the therapeutic product can be regulated, such asthrough the an administration of zinc, steroids or an agent whichaffects transcription of a protein, product or nucleic acid product oraffects the stability of a nucleic acid product.

Administration

An agent which modulates the level of expression of a Wnt protein can beadministered to a subject by standard methods. For example, the agentcan be administered by any of a number of different routes includingintravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), and transmucosal. In one embodiment, the Wntmodulating agent can be administered topically.

The agent which modulates Wnt protein levels, e.g., nucleic acidmolecules, Wnt polypeptides, fragments or analogs, Wnt modulators, andanti-Wnt antibodies (also referred to herein as “active compounds”) canbe incorporated into pharmaceutical compositions suitable foradministration to a subject, e.g., a human. Such compositions typicallyinclude the nucleic acid molecule, polypeptide, modulator, or antibodyand a pharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances are known. Except insofaras any conventional media or agent is incompatible with the activecompound, such media can be used in the compositions of the invention.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition can be formulated to be compatible with itsintended route of administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a Wnt polypeptide or anti-Wnt antibody) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active compounds are formulated into ointments, salves, gels, orcreams as generally known in the art. Such transdermal formulations canby applied to the skin to promote or inhibit hair growth.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

The nucleic acid molecules described herein can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al., PNAS 91:3054-3057, 1994). Thepharmaceutical preparation of the gene therapy vector can include thegene therapy vector in an acceptable diluent, or can include a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The agent which modulates the level of Wnt protein can be administeredby locally administration, e.g., topical administration. The agent canbe applied once or it can be administered continuously, e.g., the agentis administered with sufficient frequency such that the affect on theWnt protein level is maintained for a selected period, e.g., 5, 10, 20,30, 50, 90, 180, 365 days or more. The administration of an agent whichmodulates, e.g., increases or inhibits, the level of a Wnt protein,e.g., a Wnt polypeptide or an anti-Wnt antibody, can also be repeated.

Other Embodiments

It is understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

All patents and references cited herein are incorporated in theirentirety by reference. Other embodiments are within the followingclaims.

1. A cell culture, comprising: a dermal papilla (DP) cell, a cellculture medium, and a Wnt polypeptide or a functional fragment or analogthereof, in an amount sufficient to promote or maintain the DP cell inanagen phase.
 2. The culture of claim 1, wherein the Wnt polypeptide isWnt 3a.
 3. The culture of claim 1, wherein the Wnt polypeptide is Wnt 3.4. The culture of claim 1, wherein the Wnt polypeptide is Wnt
 4. 5. Theculture of claim 1, wherein the Wnt polypeptide is Wnt 7a.
 6. Theculture of claim 1, wherein the Wnt polypeptide is Wnt 7b.
 7. A cellculture, comprising a dermal papilla (DP) cell, a cell culture medium,and a Wnt polypeptide in an amount sufficient to promote or maintain theDP cel in anagen phase.
 8. The culture of claim 7, wherein the Wntpolypeptide is Wnt
 3. 9. The culture of claim 7, wherein the Wntpolypeptide is Wnt
 4. 10. The culture of claim 7, wherein the Wntpolypeptide is Wnt 3a.
 11. The culture of claim 7, wherein the Wntpolypeptide is Wnt 7a.
 12. The culture of claim 7, wherein the Wntpolypeptide is Wnt 7b.
 13. The cell culture of claim 1, wherein the DPcell is isolated.
 14. The cell culture of claim 2, wherein the DP cellis isolated.
 15. The cell culture of claim 3, wherein the DP cell isisolated.
 16. The cell culture of claim 4, wherein the DP cell isisolated.
 17. The cell culture of claim 5, wherein the DP cell isisolated.
 18. The cell culture of claim 6, wherein the DP cell isisolated.
 19. The cell culture of claim 7, wherein the DP cell isisolated.
 20. The cell culture of claim 8, wherein the DP cell isisolated.
 21. The cell culture of claim 9, wherein the DP cell isisolated.
 22. The cell culture of claim 10, wherein the DP cell isisolated.
 23. The cell culture of claim 11, wherein the DP cell isisolated.
 24. The cell culture of claim 12, wherein the DP cell isisolated.
 25. The cell culture of claim 1, wherein the Wnt polypeptideis recombinant.
 26. The cell culture of claim 2, wherein the Wntpolypeptide is recombinant.
 27. The cell culture of claim 3, wherein theWnt polypeptide is recombinant.
 28. The cell culture of claim 4, whereinthe Wnt polypeptide is recombinant.
 29. The cell culture of claim 5,wherein the Wnt polypeptide is recombinant.
 30. The cell culture ofclaim 6, wherein the Wnt polypeptide is recombinant.
 31. The cellculture of claim 7, wherein the Wnt polypeptide is recombinant.
 32. Thecell culture of claim 8, wherein the Wnt polypeptide is recombinant. 33.The cell culture of claim 9, wherein the Wnt polypeptide is recombinant.34. The cell culture of claim 10, wherein the Wnt polypeptide isrecombinant.
 35. The cell culture of claim 11, wherein the Wntpolypeptide is recombinant.
 36. The cell culture of claim 12, whereinthe Wnt polypeptide is recombinant.
 37. The cell culture of claim 1,wherein the Wnt polypeptide has been exogenously added to the cellculture.
 38. The cell culture of claim 2, wherein the Wnt polypeptidehas been exogenously added to the cell culture.
 39. The cell culture ofclaim 3, wherein the Wnt polypeptide has been exogenously added to thecell culture.
 40. The cell culture of claim 4, wherein the Wntpolypeptide has been exogenously added to the cell culture.
 41. The cellculture of claim 5, wherein the Wnt polypeptide has been exogenouslyadded to the cell culture.
 42. The cell culture of claim 6, wherein theWnt polypeptide has been exogenously added to the cell culture.
 43. Thecell culture of claim 7, wherein the Wnt polypeptide has beenexogenously added to the cell culture.
 44. The cell culture of claim 8,wherein the Wnt polypeptide has been exogenously added to the cellculture.
 45. The cell culture of claim 9, wherein the Wnt polypeptidehas been exogenously added to the cell culture.
 46. The cell culture ofclaim 10, wherein the Wnt polypeptide has been exogenously added to thecell culture.
 47. The cell culture of claim 11, wherein the Wntpolypeptide has been exogenously added to the cell culture.
 48. The cellculture of claim 12, wherein the Wnt polypeptide has been exogenouslyadded to the cell culture.
 49. The cell culture of claim 1, furthercomprising a cell that expresses the Wnt polypeptide. 50.The cellculture of claim 2, further comprising a cell that expresses the Wntpolypeptide.
 51. The cell culture of claim 3, further comprising a cellthat expresses the Wnt polypeptide.
 52. The cell culture of claim 4,further comprising a cell that expresses the Wnt polypeptide.
 53. Thecell culture of claim 5, further comprising a cell that expresses theWnt polypeptide.
 54. The cell culture of claim 6, further comprising acell that expresses the Wnt polypeptide.
 55. The cell culture of claim7, further comprising a cell that expresses the Wnt polypeptide.
 56. Thecell culture of claim 8, further comprising a cell that expresses theWnt polypeptide.
 57. The cell culture of claim 9, further comprising acell that expresses the Wnt polypeptide.
 58. The cell culture of claim10, further comprising a cell that expresses the Wnt polypeptide. 59.The cell culture of claim 11, further comprising a cell that expressesthe Wnt polypeptide.
 60. The cell culture of claim 12, furthercomprising a cell that expresses the Wnt polypeptide.
 61. A cell culturecomprising an isolated dermal papilla (DP) cell, cell culture medium,and a recombinant Wnt polypeptide selected from the group consisting of:Wnt 3, Wnt 3a, Wnt 4, Wnt 7a and Wnt 7b in amount sufficient to promoteor maintain the DP cell in anagen phase.
 62. A method of culturing adermal papilla (DP) cell, comprising: maintaining the cell culture ofclaim
 1. 63. A method of culturing a dermal papilla (DP) cell,comprising: maintaining the cell culture of claim 7.